Safe staphylococcal platform for the development of multivalent

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Safe staphylococcal platform for the development of multivalent nanoscale vesicles against viral infections yuan jizhen, Jie Yang, Zhen Hu, Yi Yang, Weilong Shang, Qiwen Hu, Ying Zheng, Huagang Peng, Xiaopeng Zhang, Xinyu Cai, Junmin Zhu, Ming Li, Xiaomei Hu, Renjie Zhou, and Xiancai Rao Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03893 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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Safe staphylococcal platform for the development of multivalent nanoscale

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vesicles against viral infections

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Jizhen Yuan#, Jie Yang#, Zhen Hu#, Yi Yang#, Weilong Shang#, Qiwen Hu#, Ying

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Zheng#, Huagang Peng#, Xiaopeng Zhang#, Xinyu Cai⊥, Junmin Zhu#, Ming Li#,

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Xiaomei Hu#, Renjie Zhou⊥,*, Xiancai Rao#,*

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#

Department of Microbiology, College of Basic Medical Sciences, Third Military

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Medical University, 30# Gaotanyan St., Shapingba District, Chongqing 400038, P. R.

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China.

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⊥

Department of Emergency, Xinqiao Hospital, Third Military Medical University, 83# Xinqiao St., Shapingba District, Chongqing 400037, P. R. China.

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Running title: Platform for the development of nanoscale vesicles

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ACS Paragon Plus Environment

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ABSTRACT

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Many viruses often have closely related yet antigenically distinct serotypes. An ideal

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vaccine against viral infections should induce a multivalent and protective immune

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response against all serotypes. Inspired by bacterial membrane vesicles (MVs) that

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carry different protein components, we constructed an agr locus deletion mutant of

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the Staphylococcus aureus strain (RN4220-∆agr) to reduce potential toxicity.

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Nanoscale vesicles derived from this strain (∆agrMVs) carry at least four major

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components that can deliver heterologous antigens. These components were each

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fused with a triple FLAG tag, and the tagged proteins could be incorporated into the

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∆agr

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and (2.89 ± 0.74)% of the total

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PdhA-FLAG, and Eno-FLAG, respectively. With two DENV envelope E domain III

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proteins (EDIIIconA and EDIIIconB) as models, the DENV EDIIIconA and

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EDIIIconB delivered by two staphylococcal components were stably embedded in the

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∆agr

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against all four DENV serotypes. Sera from immunized mice protected Vero cells and

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suckling mice from a lethal challenge of DENV-2. This study will open up new

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insights into the preparation of multivalent nano-sized viral vaccines against viral

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infections.

MVs. The presentation levels were (3.43 ± 0.73)%, (5.07 ± 0.82)%, (2.64 ± 0.61)%, ∆agr

MV proteins for Mntc-FLAG, PdhB-FLAG,

MVs. Administration of such engineered

∆agr

MVs in mice induced antibodies

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KEYWORDS: Staphylococcus aureus; membrane vesicle; agr; vaccine; dengue virus

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Vaccination is the most effective strategy for preventing viral diseases. However,

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many viruses often have closely related yet antigenically distinct serotypes or

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genotypes, such as poliovirus,1 herpes simplex virus,2 human papillomavirus,3 and

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dengue virus (DENV).4 Developing vaccines for diseases caused by such

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polyserotypic viruses is challenging.5 In general, the viral vaccine for each serotype or

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genotype is prepared and subsequently administrated separately or in a combined

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formulation. The development of a versatile platform for the generation of multivalent

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viral vaccines is high priority.

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Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria have been

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studied for nearly five decades and have since emerged as attractive and effective

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vaccines or delivery systems.6–9 Nano-sized OMVs can be gradually released from the

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outer membrane of Gram-negative bacteria cell walls. OMVs are also secreted from

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pathogens, such as Burkholderia pseudomallei,7 Shigella boydii,8 Salmonella enterica

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serovar Typhimurium,9 and Neisseria meningitides.10 These OMVs induce potent

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protective immune responses against certain pathogens. Moreover, OMVs from

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Gram-negative bacteria have intrinsic adjuvant roles.8,11 Several recombinant

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Meningococcus B antigens formulated with homologous strain OMVs increase

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immunogenicity.10 The serine protease HtrA of Chlamydia muridarum expressed with

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a pET21b+ vector is accumulated in the OMVs of Escherichia coli, and the resulting

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OMVs can induce neutralizing antibodies in an in vitro infectious assay, whereas the

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purified recombinant HtrA cannot.12 However, OMV vaccination is limited by the

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incorporation of lipopolysaccharide (LPS) or lipooligosaccharide (LOS) into the

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bilayer of OMVs.13 Due to the lack of outer membrane and the existence of rigid cell

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walls, direct evidence on membrane vesicle (MV) formation in Gram-positive bacteria,

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such as Staphylococcus aureus and Bacillus subtilis, was only recently demonstrated

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through transmission electron microscopic and proteomic analyses.14,15 MVs of

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Gram-positive bacteria share common features with the OMVs of Gram-negative ones;

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however, the mechanisms by which Gram-positive bacteria secrete MVs are largely

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unknown.14 Both MVs and OMVs are spherical non-replicating nanoparticles with the

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diameter of 20–200 nm and contain phospholipid bilayers that are incorporated with 3


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various bacterial proteins and lumens carrying periplasmic constituents.14,16,17

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Mycobacterium tuberculosis H37Rv MVs not only elicited protection as well as live

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BCG vaccine but also boosted BCG vaccine efficacy.18 When intraperitoneally

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injected, Streptococcus pneumoniae MVs protected mice from the homologous strain

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and other pathogenic serotype of S. pneumoniae infections.19 However, application

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studies on nanoscale MVs derived from Gram-positive bacteria to protect

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heterologous infections are not available.17

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We proposed a safe staphylococcal platform in which the toxicity of MVs was

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attenuated (∆agrMVs), and several ∆agrMV-incorporated components were characterized

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as capable of delivering heterologous viral antigens that resulted in the release of

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multivalent nanoscale ∆agrMVs against viral infections (Scheme 1). Using two DENV

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consensus peptides as model antigens, we demonstrated that the successful

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incorporation of DENV peptides into the staphylococcal

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administration of these engineered

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immune response against all the serotypes of DENV. Our data revealed the suitability

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of the staphylococcal

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MV vaccines and illustrated the potential of using this platform in developing

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multivalent viral vaccines.

∆agr

∆agr

∆agr

MVs and the

MVs induced a multivalent and protective

MV platform for developing a new generation of nanoscale

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Scheme 1. Schematic representation of the multivalent nanoscale strategy. 4


∆agr

MV generation

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Vesicle release is a ubiquitous process that occurs during normal bacterial growth

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and has been extensively characterized in Gram-negative bacteria (OMVs).20 The

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secretion of MVs from S. aureus was reported for the first time in 2009,14 and such

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report not only broke the assumption that Gram-positive bacteria could hardly release

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MVs because of the existence of thick and rigid cell walls but also provided an

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alternative to OMVs for developing potential vaccines for clinical applications.

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Taking advantage of the available genome sequences, MVs of several laboratory S.

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aureus strains (Table S1) were separately isolated, as previously described.21

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Transmission electron microscopy (TEM) revealed that all the tested S. aureus strains

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can secrete nano-sized MVs with the diameter of 49.8 ± 17.4 nm (Figures 1A and S1).

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Although the amount of vesicle released between strains has no significant difference

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(Figure 1B), the protein components that are incorporated into the staphylococcal

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MVs are strain-dependent (Figures 1C and 1D). Among the tested strains, RN4220

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has a frame shift at the 3′-terminal of the agrA gene that could delay the

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agr-controlled expression of virulence factors and may produce safe MVs.22 In

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addition, RN4220 has defects in type I restriction-modification system and is more

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convenient for performing genetic operations.23 Therefore, S. aureus RN4220 was

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selected as the starting strain in this study.

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Figure 1. Analysis of MVs derived from different S. aureus strains. (A) The MVs produced by

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S. aureus strains observed under the transmission electron microscope. The bars representing

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200 nm were indicated. (B) The production of MVs from each S. aureus strain was shown as total

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proteins (µg) in MVs derived from 1 L overnight cultures determined by the Bradford assay; n = 3

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experiments. (C) SDS-PAGE analysis of MVs derived from different S. aureus strains. The

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molecular weights of a protein marker were indicated on the left. (D) The abundance of proteins in

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the MVs of S. aureus was analyzed by the plot lane tool of ImageJ software version 1.46 (National

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Institutes of Health, USA).

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The production of S. aureus strain RN4220 MVs achieved its peak at 22 h

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post-culture (Figure 2A). Although lacking in LPS and LOS, the MVs derived from S.

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aureus could still contain some species-specific virulence factors that affect the safety

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of a potential vaccine.24 Dose-dependent experiments revealed that the survival rate

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was 0%, 10%, 20%, 50%, 80%, and 100% after BALB/c mice were intraperitoneally

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challenged with 80, 64, 51, 41, 33, and 26 µg of the wild-type RN4220-derived MVs

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(wtMVs), respectively (Figure 2B). According to the Bliss method, the calculated LD50

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for

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intraperitoneal administration. The high toxicity of

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fact that around 100 bacterial proteins are incorporated into S. aureus wtMVs,24, 25 and

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several toxic components are enriched during the MV concentration procedure.26 For

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the safety of MVs used as nano-sized vaccine delivery vehicles, characterizing every

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toxic component in wtMVs and eliminating its effect is a great challenge. Our strategy

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was to delete the whole locus of staphylococcal agr (Figure S2), a key quorum

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sensing system consisting of agrB, agrD, agrC, agrA, and RNAIII in S. aureus,27 to

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reduce the expression of virulence factors. As expected, the mice survival rate was

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100% even after they were intraperitoneally injected with 200 µg of the agr-deleted

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RN4220-derived MVs (∆agrMVs) (Figure 2B). We then examined the systemic

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inflammatory indexes IL-6 and TNFα to assess the potential toxicities of

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∆agr

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reduced after intraperitoneal injection of same dose of ∆agrMVs (Figure 2C, left panel).

wt

MVs was 2.1 mg/kg with a 95% confidence interval of 1.85–2.35 mg/kg for

MVs in vivo. Compared with

wt

wt

MVs can be explained by the

wt

MVs and

MVs, the levels of serum IL-6 were significantly 6


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However, the significantly increased TNFα level was only observed in the high-dose

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wt

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∆agr

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mouse challenged with 40 µg

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aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase

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(LDH) were also determined to further evaluate the liver toxicities of

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∆agr

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levels of ALT, AST, and LDH, whereas 20 µg

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levels compared with 100 µg

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RN4220-∆agr (∆agrMVs) exhibit attenuation in the mouse model and are safe for

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antigen delivery vehicles.

MV-challenged group (Figure 2C, right panel). Even challenged with a high dose of MVs (100 µg), mouse serum IL-6 and TNFα levels were still lower than those of wt

MVs (Figure S3). In addition, serum alanine

MVs. As shown in Figure 2D, both 20 µg and 100 µg

∆agr

wt

∆agr

wt

MVs and

MVs could not alter the

MVs significantly increased their

MVs. Taken together, MVs derived from S. aureus

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Figure 2. Deletion of agr locus attenuated the MVs of S. aureus. (A) Production of S. aureus

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RN4220

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BALB/c mice (6–8 weeks old) challenged with wtMVs (80, 64, 51, 41, 33, and 26 µg) and ∆agrMVs

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(200 µg) (n = 10 for each group). (C) Mouse serum levels of IL-6 and TNFα 6 h

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post-intraperitoneal injection of wtMVs (10, 20, and 40 µg) and ∆agrMVs (10, 20, and 40 µg) (n = 4

wt

MVs during different growth stages (n = 3 for each time point). (B) Survival rates of

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wt

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for each dose). (D) Hepatic injury indexes after intraperitoneal injection of

MVs (20 µg) and

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∆agr

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group). Data are presented as the mean ± SD. n.s. represented no significance, * P < 0.05, **

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P < 0.01, and *** P < 0.001.

MVs (20 µg, 100 µg). CCl4 (10% in corn oil) was used as a positive control (n = 4 for each

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SDS-PAGE revealed that the protein patterns of RN4220-∆agr and wild-type

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∆agr

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RN4220 were very similar; however, the protein components of

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some extent compared with those of the

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performed a proteomic analysis to explain why the agr locus deletion reduced the

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toxicity of MVs. A total of 92 proteins were identified in the

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components were characterized in the ∆agrMVs (Table S2). wtMVs and ∆agrMVs had 61

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common proteins (Figure 3B). This phenomenon has also been observed in a recent

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study where the MVs from three S. aureus isolates carried 25 common proteins and

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60 strain-speciﬁc proteins.28 Proteomics analysis also revealed that the most abundant

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protein in

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modified protein abundance index (emPAI) value of 93876.4, followed by PSMα

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(2259 Da, emPAI = 6.9) and alpha-hemolysin (emPAI = 1.02) (Figure 3C). However,

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these virulence factors were absent in the

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Table S2), thereby supporting the survival rate of the ∆agrMV-challenged mouse model

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(Figure 2B). Gene Ontology (GO) analysis revealed that the major components in

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∆agr

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metabolic process with catalytic activity (Figure S4). Moreover, Keiser et al.29

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reported that detoxifying the LOS of OMVs by disabling the lpxL2 gene in group B

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Meningococcus results in reduced viability of the bacteria; however, deletion of agr

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locus in S. aureus (RN4220-∆agr) would not affect bacterial growth (Figure S5A),

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and

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RN4220-∆agr (Figure S5B), thereby indicating a successfully engineered host of S.

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aureus RN4220-∆agr for the production of nanoscale vesicles.

wt

MVs varied to

MVs (Figure 3A). We subsequently

wt

MVs, and 119

wt

MVs was delta-hemolysin (5009 Da), which had an exponentially

∆agr

MVs from RN4220-∆agr (Figure 3D,

MVs were cytosolic and cytoplasmic proteins that are involved in the cellular

∆agr

MV protein patterns were similar after logarithmic growth of the engineered

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Figure 3. Proteomic analysis of proteins incorporated in wtMVs and ∆agrMVs. (A) Comparison

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of protein profiles between S. aureus RN4220 and RN4220-∆agr, as well as their derived MVs

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(wtMVs and ∆agrMVs). The sizes of a protein marker were indicated on the left. The major bands of

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∆agr

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indicated by a black arrow. (B) Proteins detected in the proteomic analysis with liquid

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chromatography-tandem mass spectrometry (LC-MS/MS) in the MVs derived from S. aureus

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RN4220 and RN4220-∆agr. The number (percent) of the identical and different proteins in the

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wt

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protein in wtMVs was delta-hemolysin with an emPAI value of 93876.4, followed by PSMα and

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alpha-hemolysin, as indicated. (D) Major components detected in

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The major components in ∆agrMVs seemed scattered with the most predominant protein of enolase

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(47,177 Da, emPAI = 3.4).

MVs were indicated by black triangles, and the abundant band that was absent in ∆agrMVs was

MVs and ∆agrMVs was indicated. (C) Major components detected in wtMVs. The most abundant

∆agr

MVs with the emPAI > 1.

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The major components that corresponded to the rich bands in SDS-PAGE gel were

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shared by wtMVs and ∆agrMVs (Figure 3A). Eight abundant bands with variable sizes

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in the

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by LC-MS/MS to select the optimal components that can deliver heterologous

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antigens. Seven of these proteins were also identified in the proteomic analysis of

∆agr

MV gel (Figure 3A, indicated by black triangles) were further characterized

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wt

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potential capacity of these proteins in delivering heterologous antigens, a DNA

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sequence encoding 3×FLAG tag (DYKDHDGDYKDHDIDYKDDDDK) was

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chemically synthesized and genetically in-frame fused with the 3′-terminal of each

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protein encoding gene of S. aureus RN4220-∆agr through homologous recombination.

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Four

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RN4220-∆agr/Mntc-FLAG, RN4220-∆agr/PdhB-FLAG, RN4220-∆agr/PdhA-FLAG,

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and RN4220-∆agr/Eno-FLAG (Figure S6). These fusion proteins could be detected in

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the knocked-in strains and incorporated into the ∆agrMVs by using mAb against FLAG

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(Figures 4A and 4B). Quantitative Western blot showed that the FLAG-fused proteins

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represented (3.43 ± 0.73)%, (5.07 ± 0.82)%, (2.64 ± 0.61)%, and (2.89 ± 0.74)% of

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the total

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Eno-FLAG, respectively (Figures 4C and S7). However, the reasons for the failure of

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RplE (20.3 kDa), PdhC (46.3 kDa), and GlnA (50.9 kDa) in carrying heterologous

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antigens remained unclear. The failure might either be due to the technical problems

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or the gene itself for S. aureus survival. At least four abundant components with

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variable sizes in

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∆agr

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attenuating toxicity while possessing a heterologous antigen delivery capability.

MVs and

∆agr

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MVs, and their emPAI values were above 1.0 (Table S3). To test the

knocked-in

strains

were

successfully

constructed,

designated

as

∆agr

MV proteins for Mntc-FLAG, PdhB-FLAG, PdhA-FLAG, and

∆agr

MVs are capable of delivering heterologous antigens into the

MVs, thereby highlighting the staphylococcal platform as an effective mode of

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Figure 4. Characterization of protein components’ potential for delivery of heterologous

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antigens. (A) Western blot analysis of 3×FLAG-tagged proteins in the total cell lysates using

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anti-FLAG monoclonal antibodies. The full-length blots are presented in Figure S10. (B) Western

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blot analysis of 3×FLAG-tagged proteins in total

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Experimental section. (C) Abundance of FLAG-tagged protein in the

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of

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and analyzed by the plot lane tool of the ImageJ software version 1.46 (right panel). Certain

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FLAG-tagged proteins were detected by Western blot (central panel); their amounts were

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determined using a standard curve from the Western blot for pure Eno-FLAG in

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S7) and indicated on the top as a percentage to the total

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corresponding to the band in gel was indicated by a black triangle.

∆agr

MVs prepared as described in the ∆agr

MVs. The total proteins

∆agr

MVs derived from each knocked-in strain were separated by 12% SDS-PAGE (left panel)

∆agr

MVs (Figure

∆agr

MV proteins. The relative peak

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To test the validity of staphylococcal platform for the generation of multivalent

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∆agr

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DENV serotypes were selected as model antigens. DENV has four serotypes, which

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were designated as DENV-1, -2, -3, and -4. These serotypes are widely distributed in

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tropical and subtropical regions that affect approximately 3.6 billion people

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worldwide.30 Previous reports indicated that EDIII is the major protective domain for

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prevention of DENV infections.31,32 Among the four DENV serotypes, the EDIIIs of

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DENV-1 and -3 are the most similar to each another, whereas DENV-2 is the closest

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to DENV-4.33 To simplify the construction process, physicochemical property method

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(PCP method)33,34 was used to generate two PCP-consensus EDIII sequences

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representing DENV-1/-3 (EDIIIconA) and DENV-2/-4 (EDIIIconB). Four EDIII

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PCP-consensus sequences (DENV-1–4con, Figure S8A) were first created based on

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the individual alignments of 356 DENV-1, 147 DENV-2, 146 DENV-3, and 181

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DENV-4 sequences derived from GenBank (http://www.ncbi.nlm.nih.gov). Then,

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EDIIIconA was obtained by PCP method using DENV-1con and DENV-3con as

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inputs, analogously to obtain EDIIIconB that represents the DENV-2/-4 (Figure S8B).

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The coding sequences of EDIIIconA and EDIIIconB were deduced according to

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the codon usage bias of S. aureus RN4220 (Figure S8C) and were chemically

MVs, the envelope E protein domain III (EDIII, residues 296–395) of different

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synthesized to decrease the effect of codon bias on the expression of target fusion

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protein. Then, EDIIIconA and EDIIIconB sequences were in-frame fused with the

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3′-terminal of eno and pdhB genes in the genomic DNA of S. aureus RN4220-∆agr,

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respectively (Figure 5A). The expected productions of Eno-EDIIIconA and

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PdhB-EDIIIconB fusion proteins were confirmed by Western blot analysis in both

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total cell lysates of the engineered bacteria and the total ∆agrMVs (Figures 5B and 5C).

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Immunoelectron microscopy (IEM) revealed that DENV EDIII antigens showed

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efficient labeling using anti-EDIII and were mainly carried in the lumen or presented

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on the surface of

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can be delivered into the

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vesicular components of S. aureus RN4220-∆agr.

∆agr

MVs (Figures 5D and 5E). The viral antigen (dengue EDIIIcon) ∆agr

MVs through the orientation of characterized major

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Figure 5. Expression and identification of the Eno-EDIIIconA and PdhB-EDIIIconB fusion

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proteins. (A) Schematic representation of the size changes when Eno and PdhB fused with

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EDIIIconA and EDIIIcinB, respectively. (B) Western blot analysis of target fusion proteins in the

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total cell lysates of RN4220-∆agr and RN4220-∆agr-EDIIIconA/B using anti-Eno antibodies (left

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panel), anti-PdhB antibodies (central panel), and anti-DENV-2con antibodies (right panel). The

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full-length blots are presented in Figure S10. (C) Western blot analysis of fusion proteins in the

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total

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panel), anti-PdhB antibodies (central panel), and anti-DENV-2con antibodies (right panel). (D)

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Immunoelectron micrographs of EDIIIconA/B contained

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Experimental section. Micrographs show efficient labeling of EDIIIconA/B-fused proteins with

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mouse-anti-DENV-2con antibodies (indicated by black arrows). (E) Immunoelectron micrographs

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of ∆agrMVs derived from RN4220-∆agr served as negative control.

∆agr

MVs of RN4220-∆agr and RN4220-∆agr-EDIIIconA/B using anti-Eno antibodies (left

∆agr

MVs detected, as described in the

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Given the potent component-mediated delivery of viral antigens into the ∆agrMVs,

302

we speculated that engineered dengue ∆agrMVs can evoke host immune responses. The

303

EDIIIconA/B contained multivalent ∆agrMV-induced higher titers of antibodies against

304

the recombinant EDIII (rEDIII) proteins of all four DENV serotypes than the

305

EDIIIconB contained

306

multivalent

∆agr

MVs

∆agr

MVs did (Figure 6). With only prime vaccination, the

induced

higher

binding

antibodies

13


than

the

rEDIII

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Page 14 of 21

∆agr

307

protein-challenged (Figure 6A). EDIIIconB contained

308

antibodies against the rEDIII of DENV-2 and DENV-4. Similar results were observed

309

in the prime-boost vaccination schedule (Figures 6B and 6C), which were concordant

310

with the fact that EDIIIconB (representing DENV-2/-4) contains less epitopes against

311

DENV-1 and DENV-3. In the three prime-boost schedules, EDIIIconA/B contained

312

multivalent

313

serotypes of DENV regardless of the use of adjuvants (Figures 6B and 6C), thereby

314

suggesting that staphylococcal ∆agrMVs may have an adjuvant effect similar to that of

315

the OMVs derived from Gram-negative bacteria.8,11 The engineered ∆agrMVs carrying

316

dengue EDIII antigens can effectively evoke humoral immune responses in mice with

317

intrinsic adjuvant activity. However, the mechanism by which staphylococcal ∆agrMVs

318

display adjuvant capacity requires further investigation.

∆agr

MV-evoked higher binding

MV-induced similar humoral immune responses against all the four

319 320

Figure 6. Capture ELISA detected the binding antibodies in mouse sera against recombinant

321

EDIII (rEDIII) antigens (DENV-1–4con). (A) Sera derived from immunized mice with a single

322

vaccination of EDIIIconA/B or EDIIIconB ∆agrMVs without adjuvants. Sera from mice challenged

323

with DENV-1–4con (rEDIII) and PBS served as positive and negative controls, respectively. (B) 14


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Nano Letters

324

Sera derived from immunized mice with thrice prime-boost schedule of EDIIIconA/B or

325

EDIIIconB

326

prime-boost schedule of EDIIIconA/B or EDIIIconB

327

arbitrary units. Data represent three independent experiments and expressed as mean ± SD.

∆agr

MVs without adjuvants. (C) Sera derived from immunized mice with thrice ∆agr

MVs with Freund’s adjuvants. AU,

328 329

To test whether the immunized mouse sera could neutralize virus infection, plaque

330

reduction neutralization test (PRNT)35 was performed. The neutralizing effect of

331

EDIIIconA/B contained

332

dose-dependent. The 320 times diluted immune sera could still inhibit approximately

333

75% formation of DENV-2 plaques, whereas the control sera at 1:40 dilution could

334

only offer an inhibition of less than 20% (Figure 7A). Immunofluorescence assay

335

(IFA) was also used to monitor the multiplication of DENV-2 in Vero cells after

336

neutralizing the virus with immunized murine sera against EDIIIconA/B multivalent

337

∆agr

338

DENV-2 infection, whereas the normal BALB/c mouse sera could not (Figure 7B).

339

These results were consistent with those of PRNT (Figure 7A). Furthermore, the

340

protective activity of the immunized sera was also evaluated in vivo. Approximately

341

80% of the tested suckling mice were protected from the lethal dose challenge of

342

DENV-2, which was pre-incubated with 1:80 diluted immunized murine sera. All the

343

control mice died 10 days after they were post-challenged by normal sera pretreated

344

virus (Figure 7C). EDIIIconA/B-loaded ∆agrMVs can induce dengue-specific humoral

345

immune responses and confer effective protection against DENV-2 infection.

∆agr

MV-immunized mouse sera (three prime-boost) was

MVs. The immunized sera diluted at 1:80 could completely protect Vero cells from

15


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Page 16 of 21

346 ∆agr

347

Figure 7. Protective roles of EDIIIconA/B multivalent

MV sera against DENV-2. (A)

348

Neutralizing activities of immunized sera against DENV-2 determined by plaque reduction

349

neutralization test (PRNT). Diluted sera from immunized mice treated thrice with prime-boost of

350

EDIIIconA/B

351

cells. Results were expressed as the percentage blocking (PB%) in PRNT. PB% was calculated as

352

described in the Experimental section. Normal mouse sera served as negative control. (B)

353

Multiplication of DENV-2 in Vero cells tested with an immunofluorescence assay (IFA). The

354

dilutions of sera from the EDIIIconA/B

355

were used as negative control. (C) Survival rates of BALB/c suckling mice challenged with

356

DENV-2 pre-incubated with 1:80 diluted sera from EDIIIconA/B

357

circle) or with sera from normal mice (filled square).

∆agr

MVs without adjuvants were incubated with DENV-2 before infection to Vero

∆agr

MV-immunized mice were indicated, and normal sera

∆agr

MV-immunized mice (filled

358 359

In conclusion, the applicability of the major components of S. aureus MVs for

360

delivering serotypic viral antigens as multivalent nanoscale vaccines was tested. We

361

proposed an agr-deleted S. aureus strain with four well-characterized MV components

362

for the delivery of heterologous antigens to produce multivalent vaccines. This safe 16


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Nano Letters

363

platform has a broad range of biotechnological applications, including the generation

364

of multivalent vaccines against viruses with several serotypes and/or different viruses

365

(Figure S9). However, this platform has some limitations. It may not be particularly

366

useful for expressing glycosylated antigens. The protective viral peptides without

367

glycosylation, such as DENV EDIIIs, are satisfied. In addition, the production of

368

∆agr

369

a reasonable scale for pharmaceutical applications, and further biotechnological

370

optimization is needed. Our study provides an innovative attempt to use

371

Gram-positive bacteria-released nanoscale MVs as immunologic tools for fighting

372

viral infections.

MVs using engineered RN4220-∆agr may rarely be sufficiently efﬁcient to achieve

373 374 375 376

ASSOCIATED CONTENT

377

Supporting Information

378

Detailed description of the Experimental section and additional data are provided.

379

Figures S1–S10, Tables S1–S5.

380 381

AUTHOR INFORMATION

382

Corresponding Authors

383

*X. R. ([email protected]); R. Z. ([email protected]).

384 385

Author Contributions

386

X.R., R.Z., and X.H. conceived and designed the experiments. J.Y., Z.H., W.S., Y.Z.,

387

H.P., X.C., and X.Z. performed the experiments. J.Y., X.R., Q.H., M.L., J.Z., and Y.Y.

388

analyzed the data. J.Y., Y.Y., and X.R. wrote the manuscript. All authors discussed the

389

results and commented on the manuscript. The principal investigator is X.R. and R.Z.

390 391

ORCID

392

Jizhen Yuan, 0000-0003-4693-9534; Jie Yang, 0000-0002-2886-9774; Zhen Hu, 17


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Yang,

0000-0002-3707-5052;

Page 18 of 21

393

0000-0002-1077-6991;

Yi

394

0000-0002-0752-5016;

Qiwen

395

0000-0002-9481-2733; Huagang Peng, 0000-0002-3482-0213; Xiaopeng Zhang,

396

0000-0002-3880-1810;

Xinyu

Cai,

397

0000-0002-1652-5421;

Ming

Li,

398

0000-0003-2831-1241;

Renjie

Zhou,

399

0000-0002-9905-760X.

Hu,

Weilong

Shang,

Ying

Zheng,

0000-0001-6689-6194;

0000-0002-3045-6899; 0000-0003-1662-6465; 0000-0003-3046-567X;

Junmin

Zhu,

Xiaomei

Hu,

Xiancai

Rao,

400 401

Notes

402

The authors declare no competing financial interests.

403 404

ACKNOWLEDGMENTS

405

We thank Prof. Daoguo Zhou (Purdue University) for critical reading of the

406

manuscript. This work was supported by the national natural science foundation of

407

China (grant no. 31270979 to X.R.), the new drug development project of China

408

(grant no. 2012ZX09103301-038 to X.R.) and the translational project of TMMU

409

(grant no. 2016XZH01 to X.R.). The funders had no role in study design, data

410

collection and analysis, decision to publish or preparation of the manuscript.

411 412 413

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Safe staphylococcal platform for the development of multivalent

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