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com/science/article/pii/S0169409X22001843
Manuscript_9d799fa136bb29858bcf298dee15b0f6

Bacterial membrane vesicles for vaccine applications

Nishta Krishnan, Luke J. Kubiatowicz, Maya Holay, Jiarong Zhou, Ronnie H. Fang*, and

Liangfang Zhang*

Department of NanoEngineering, Chemical Engineering Program, and Moores Cancer Center,

University of California San Diego, La Jolla, CA 92093, U.S.A.

*Corresponding authors: [email protected] (R.H. Fang), [email protected] (L. Zhang)

© 2022 published by Elsevier. This manuscript is made available under the Elsevier user license
https://www.elsevier.com/open-access/userlicense/1.0/
Abstract

Vaccines have been highly successful in the management of many diseases. However,

there are still numerous illnesses, both infectious and noncommunicable, for which there are no

clinically approved vaccine formulations. While there are unique difficulties that must be

overcome in the case of each specific disease, there are also a number of common challenges that

have to be addressed for effective vaccine development. In recent years, bacterial membrane

vesicles (BMVs) have received increased attention as a potent and versatile vaccine platform.

BMVs are inherently immunostimulatory and are able to activate both innate and adaptive

immune responses. Additionally, BMVs can be readily taken up and processed by immune cells

due to their nanoscale size. Finally, BMVs can be modified in a variety of ways, including by

genetic engineering, cargo loading, and nanoparticle coating, in order to create multifunctional

platforms that can be leveraged against different diseases. Here, an overview of the interactions

between BMVs and immune cells is provided, followed by discussion on the applications of

BMV vaccine nanotechnology against bacterial infections, viral infections, and cancers.

Keywords: biomimetic, vaccination, immunotherapy, bacterial membrane vesicle,

nanotechnology

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1. Introduction

Vaccination strategies have been incredibly successful at combatting a number of deadly

diseases, helping to prevent their spread across the global population [1]. The power of vaccines

has further been highlighted by the current pandemic, where formulations against the severe acute

respiratory syndrome coronavirus 2 (SARS-CoV-2) have proven highly effective [2]. With the

ever-present threat of emerging pandemics, as well as the growing incidence of other vaccinatable

diseases such as cancer, the need for continued development on potent vaccine formulations is

apparent. Researchers are continually searching for novel platform technologies with wide

applicability, simple production schemes, and the ability to achieve strong immune potentiation.

Recently, bacterial membrane vesicles (BMVs) have emerged as useful components that can be

incorporated into new vaccine formulations [3]. These naturally occurring nanostructures have

many useful biological functions that can be leveraged for immune-related applications [4-6]. In

nature, BMVs can hold and distribute nutrients, virulence factors, and toxins while also depleting

antibacterial molecules generated by the host [7, 8]. Additionally, due to their biogenesis from the

surface of bacteria, BMVs display a wide range of bacterial membrane antigens that can serve as

prime vaccine targets [9]. Generally between 50-200 nm in size, BMVs have suitable dimensions

that allow them to be readily taken up and processed by immune cells. As they contain a high

density of pathogen-associated molecular patterns (PAMPs) that serve as strong immune

potentiators, BMVs are naturally self-adjuvanting and effective drivers of the innate immune

system. Several studies have highlighted the ability of BMVs to induce long-lasting humoral and

cellular immune responses when used as vaccines [10-12].

Compared to traditional vaccination approaches, BMVs show great promise due to their

simplicity of production, broad applicability, and self-adjuvanting properties. As such, BMVs

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have emerged as an attractive platform for vaccine development [13-16]. Research over the last

decade has demonstrated BMVs as powerful, economical, and flexible tools for the delivery of

various vaccine antigens, including those from bacterial, viral, and even cancer sources (Fig. 1).

In this review, we first introduce BMVs and various approaches for their production and

functionalization. We then discuss the activation of the immune system by these naturally

occurring bacterial vesicles, as well as their notable vaccine-related applications.

2. Overview of BMVs

Bacteria release membrane vesicles that are capable of various biological functions,

including exerting virulence, transferring genetic material, and modulating cellular signaling

[17]. With these abilities, BMVs represent a powerful platform that can be leveraged to treat and

prevent a wide range of diseases. Understanding the structure, biogenesis, and opportunities for

modification of BMVs enables researchers to better apply these naturally occurring

nanostructures towards biomedical applications.

2.1. Structure and composition

Ranging from 20-400 nm, BMVs are natural transporters that carry a wide range of

cargoes, including lipopolysaccharide (LPS), peptidoglycans, lipids, periplasmic and cytoplasmic

proteins, toxins, and nucleic acids [18]. With the differences in architecture between Gram-

negative and Gram-positive bacteria, there are variances in the biogenesis and composition of

their respective BMVs. Gram-negative bacteria are characterized by a cellular envelope that

consists of three distinct layers [19]. The innermost is a fluid phospholipid bilayer, while the

middle layer is a peptidoglycan cell wall comprised of covalently crosslinked macromolecules.

Finally, the outermost layer is a phospholipid membrane containing LPS. This outer membrane

4
encases the BMVs as they are produced by Gram-negative bacteria [20]. BMVs from Gram-

negative bacteria can be produced through blebbing from the outer membrane, or spontaneous

cell lysis in which membrane fragments from lysed bacteria reform around cellular components

[18]. Gram-positive bacteria are surrounded by a thick and rigid cell wall outside of their

cytoplasmic membrane layer [19]. In Gram-positive bacteria, BMVs are thought to be released

via the enzymatic degradation of the rigid cell wall, which results in the generation of pores

through which membrane vesicles can protrude [18]. Similar to those from Gram-negative

bacteria, Gram-positive BMVs contain a variety of proteins, nucleic acids, and toxins, but they

lack an outer membrane coating.

2.2. Unmodified BMVs

Upon being secreted from bacteria, BMVs must be isolated before they can be further

used. Traditional isolation approaches include ultracentrifugation, filtration, and precipitation

[21]. While the ultracentrifugation of bacterial culture supernatant remains a widely used process

to collect BMVs, it faces many challenges, including low yields, larger and varied vesicle sizes,

and poor scalability. To isolate BMVs by filtration, culture supernatant is passed through a

porous membrane with a given molecular weight cutoff, generally within the range of 50 to 100

kDa [22]. Through this process, proteins that are not associated with the BMVs are removed.

Filtration techniques generally offer consistent yields and low batch-to-batch variability, but are

unable to fully remove large protein aggregates and other contaminants [21]. Finally, in

precipitation-based methods of isolation, high concentrations of salts such as ammonium sulfate

are added into the culture supernatant to modify the surface charges of BMVs [23]. This reduces

the colloidal stability of the vesicles, thus causing them to aggregate, which allows for facile

separation by conventional centrifugation. While straightforward and scalable, the precipitation

5
process can potentially damage important proteins while also co-precipitating soluble protein

contaminants. Many BMVs isolated by these traditional methods have been developed as

vaccines, including some against Acinetobacter baumannii, Bordetella pertussis, Staphylococcus

aureus, and Neisseria meningitidis, among others [24-27].

While basic isolation processes can be employed to purify out many toxins and harmful

components of bacteria, toxicity and safety remain a key concern when using BMVs [21]. As

such, further purification to remove larger extracellular materials such as flagella, pili, and protein

aggregates is highly important. Along these lines, more sophisticated purification techniques have

been employed. Due to their high lipid content, BMVs commonly have a lower density compared

to soluble proteins that are secreted by bacteria, enabling them to be effectively processed by

density gradient centrifugation [28]. After centrifugation, individual fractions can be collected and

analyzed to identify the precise location of the BMVs. In cases where high purity and size

homogeneity are an important consideration, filtration techniques such as gel sieving or size

exclusion chromatography can provide a suitable approach. However, the application of these

techniques oftentimes comes at the cost of reduced yield. BMV vaccine production can involve a

combination of the techniques described above. In an example, Escherichia coli culture

supernatant was first passed through a 100 kDa filtration device, followed by centrifugation on an

iodixanol density gradient (Fig. 2a) [29]. Analysis of the derived BMVs showed retention of

surface proteins through the isolation and purification process.

2.3. Cargo-loaded BMVs

The incorporation of payloads into or onto BMVs can improve their pharmacological

properties. Several approaches for cargo loading have been developed, including encapsulation

into the interior of the BMVs, or conjugation onto the exterior through surface modification

6
approaches [30]. BMVs can be functionalized either after isolation or during biogenesis, with

each methodology providing certain advantages.

As they contain a lipid bilayer structure, BMVs are capable of carrying both hydrophobic

and hydrophilic compounds. Incorporation of payloads after the isolation and purification of

BMVs from culture can be accomplished in a variety of ways. Passive methods such as

diffusion, including along chemical gradients, is a common approach for cargo loading [31].

Passive loading is suitable for positively charged molecules that are drawn to the negatively

charged membrane surface, as well as hydrophobic compounds. However, negatively charged

compounds cannot be loaded efficiently into BMVs through purely passive approaches. In such

cases, techniques such as electroporation or ultrasonication can be used to generate pores in the

BMVs and allow for payload passage through the membrane [30]. Cell-penetrating peptides and

membrane-destabilizing chemicals can also be used to enhance membrane permeability and

allow for cargo entry. In an example of membrane destabilization, electroporation was used to

generate pores in BMVs derived from Pseudomonas aeruginosa, allowing gold nanoparticles 10

nm in size to enter (Fig. 2b) [32].

Functionalization of BMVs during biogenesis can be accomplished through incubation of

payloads with bacteria cultures. The bacteria engulf the molecule of interest, which is then

packaged into any BMVs that are subsequently produced [30]. This approach was leveraged to

load antibiotics into BMVs derived from P. aeruginosa [33]. Because the bacteria are resistant to

aminoglycosides, these antibiotics could be effectively packaged into the secreted BMVs for the

treatment of aminoglycoside-sensitive infections. It is important to note that this approach can be

used as long as the resistance of the source bacteria is not mediated by the enzymatic degradation

of the antibiotic molecule.

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2.4. Genetically modified BMVs

A number of molecular biology techniques have been developed to genetically modify

bacteria for a variety of purposes. As such, there is significant potential to improve BMVs

through the genetic manipulation of their source bacteria. Genetic engineering can be employed

to reduce toxicity, improve yield, improve targeting to disease sites or cells of interest, and

express antigens [22].

Particularly in the case of BMV vaccines derived from Gram-negative bacteria, the

presence of endotoxins is a major concern. Genetic manipulation of the parent bacteria to modify

endotoxin production is a potential avenue for generating safer vaccine platforms. In a BMV-

based vaccine derived from N. meningitidis, toxicity was reduced by modifying genes related to

the production of LPS [34]. The genetically modified BMVs were still able to elicit dendritic cell

maturation, indicating their potential for vaccine applications. This formulation was further

engineered to increase BMV yield via the deletion of the rmpM gene [35]. Bacteria with RmpM

deletions display loosely attached outer membrane, allowing for easier release of BMVs and

increased yield. Another approach to increase the yield of BMVs is through modulation of the

Tol-Pal system [22]. The Tol-Pal system is comprised of 5 proteins in the envelope of Gram-

negative bacteria and is essential for maintaining the integrity of the bacterial outer membrane

[36]. Introduction of a mutant tolB gene resulted in increased OMV release in E. coli, Salmonella

enterica serovar Typhimurium, and Helicobacter pylori [37-39].

Genetic engineering can also be leveraged to introduce antigenic epitopes onto the BMV

surface. Such an approach was used to display an N. meningitidis antigen, NspA, onto the

surface of the commensal Neisseria flavescens [40]. The NspA-expressing BMVs derived from

these engineered bacteria were able to confer protection against a lethal challenge using N.

8
meningitidis. Another method of introducing epitopes is to recombinantly fuse protein sequences

to native proteins that are naturally expressed on the surface of BMVs [22]. This concept was

demonstrated in E. coli by genetically fusing proteins to cytolysin A (ClyA), which is naturally

found on the surface of E. coli BMVs [41]. Taking advantage of this process, functional

enzymes, fluorescent proteins, and antibodies were successfully expressed. This approach was

also leveraged to introduce functional molecules onto the surface of S. enterica BMVs [42]. A

similar engineering mechanism was developed using the autotransporter (AT) pathway, which

leverages a translocation signal for expression on the outer membrane [43]. Genetic fusion to an

AT protein allowed for the presentation of heterologous proteins on the surface of E. coli and

Salmonella Typhimurium BMVs [44]. Non-native anchoring proteins can also be exploited to

engineer epitopes onto the surface of BMVs through post-production binding. A notable example

is the use of SpyTag and SpyCatcher, a protein-based pair designed to form covalent bonds with

each other [45]. In an example, SpyTag was expressed on the surface of E. coli BMVs, while the

enzyme phosphotriesterase was genetically conjugated to SpyCatcher (Fig. 2c) [46]. Through the

covalent linking of SpyTag and SpyCatcher, phosphotriesterase was immobilized onto the

surface of the BMVs.

2.5 BMV-coated nanoparticles

In recent years, cell membrane coating nanotechnology has established itself as a versatile

and powerful approach for nanoparticle functionalization [47]. As the outermost layer of the cell,

the membrane is responsible for many interactions with the surrounding environment. By taking

this outermost layer and transferring it to surface of a nanoparticle core, the resulting membrane-

coated nanoparticle exhibits many properties of the parent cell. First conceptualized using red

blood cell membrane to coat polymeric nanoparticles [48], this technology has since been

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generalized to utilize the membrane from platelets, macrophages, T cells, neutrophils, endothelial

cells, and stem cells for a diverse range of applications, including detoxification, drug delivery,

immune manipulation, and theranostics [49-54]. Recent works have shown the potential of using

cell membrane-coated nanoparticles, including those derived from bacteria, as a platform for

vaccination [55-59]. In contrast to free BMVs, the size of BMV-coated nanoparticles can be more

readily controlled, and the core can be loaded with a wider range of payloads. This enables them

to more effectively manipulate the immune system for vaccine applications.

In one case, BMVs derived from E. coli were coated onto gold nanoparticles with a

diameter of 30 nm through a co-extrusion procedure [57]. The resulting BMV-coated

nanoparticles were able to induce potent dendritic cell maturation and significantly elevated the

production of proinflammatory cytokines, demonstrating their potential as a vaccine against E.

coli. A similar formulation leveraged BMVs derived from H. pylori to coat polymeric

nanoparticles through a facile sonication process (Fig. 2d) [58]. In another example, BMVs

derived from S. aureus were coated onto polymeric cores preloaded with either vancomycin or

rifampicin [60].

3. Activation of the immune system by BMVs

Over the course of their evolution, bacteria have adapted to survive and proliferate in

their host environment [61]. In parallel, the mammalian immune system has evolved mechanisms

that are responsible for recognizing and eradicating invading pathogens (Fig. 3). Various PAMPs

from bacteria, including those based on proteins, lipids, glycans, and nucleic acids, can bind to

different families of pattern recognition receptors (PRRs) present on host cells, initiating a strong

immune response (Table 1) [62, 63]. Originating from bacteria, BMVs share many of the same

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immunomodulatory features, enabling them to interact and interface with different immune cell

populations and thus generate potent immune responses [17].

Against bacteria, the first step in the activation of the immune system occurs as a result of

the interaction between PAMPs and PRRs on innate immune cells [64]. Among the PRRs, Toll-

like receptors (TLRs) play a vital role in the response against BMVs [65]. TLRs are expressed by

innate immune cells such as dendritic cells (DCs) and macrophages, as well as by non-immune

cells such as fibroblasts and epithelial cells. Cell surface TLRs mainly recognize microbial

membrane components such as lipids, lipoproteins, and proteins [65]. Notable cell surface TLRs

include TLR4 and TLR5, which recognize bacterial lipopolysaccharides and flagellin,

respectively [66]. Others include TLR1, TLR2, and TLR6, which together can recognize a wide

variety of PAMPs, including lipoproteins, peptidoglycans, lipotechoic acids, zymosan, mannan,

and tGPI-mucin [65]. Intracellular TLRs are responsible for recognizing bacterial and viral

nucleic acids, as well as self-nucleic acids resulting from disease conditions such as

autoimmunity. Engagement of TLRs by PAMPs results in increased proinflammatory cytokine

and chemokine production via the nuclear factor-κB (NF-κB) signaling pathway and TIR-

domain-containing adapter-inducing interferon-β (TRIF) signaling pathway [67].

Besides the TLR family, other receptors also participate in the recognition of bacterial

PAMPs, including nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), a

class of cytoplasmic PRRs [62]. One subclass of NLRs are the NOD receptors, which include

NOD1 and NOD2 that recognize the bacterial peptidoglycan components, diaminopimelic acid

and muramyl dipeptide, respectively [68]. These NLRs activate NF-κB, mitogen-activated protein

kinases (MAPKs), and interferon (IFN) regulatory factors [69]. Another subclass of NLRs is the

NLRP receptors, NLRP1 and NLRP3, which are characterized by the presence of a pyrin domain

11
that can activate the inflammasome pathway [70]. NLRP receptors are activated by PAMPs such

as bacterial toxins. Finally, it has been seen that the presence of cytosolic DNA can trigger

cytokine secretion via the stimulator of interferon genes (STING) pathway [71]. Cytosolic DNA

activates host cyclic GMP-AMP synthase, inducing the production of cyclic dinucleotides, which

engage STING to activate NF-κB and interferon regulatory factor 3 (IRF3) signaling. Other PRRs

include the retinoic acid-inducible gene 1-like receptors and the C-type lectin receptors, which

play a large role in the detection of viral and fungal pathogens, respectively [72].

The interactions between PRRs and PAMPs lead to the activation and maturation of

antigen-presenting cells (APCs) through different signaling pathways, and this is essential for the

transition from innate to adaptive immunity [62]. NF-κB, MAPK, and inflammasome activation

results in the upregulation of proinflammatory cytokines, chemokines, and additional

inflammatory mediators in APCs such as DCs and macrophages [67]. Upon engagement with

APCs presenting their cognate antigen via major histocompatibility complexes (MHCs), naïve T

cells are activated and differentiate into different subsets [73]. Importantly, active helper T cells

produce proinflammatory cytokines and chemokines, and they interface with B cells to stimulate

antibody production [74]. Inflammasome activation during infection has been shown to enhance

the proliferation and survival of effector CD8+ T cells and induce IFN-γ production from

memory cytotoxic T lymphocytes [75]. Overall, the priming of the adaptive immune system

allows for a concerted and specific response to clear active infections, while also protecting

against future exposures.

4. Vaccine applications of BMVs

4.1 Bacterial infections

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 Bacterial infections are commonly managed therapeutically with antibiotics that target

various biochemical mechanisms. Despite the success of this strategy since the discovery of

penicillin in the early 1900s [76], the direct selection pressure that is exerted on bacteria by

antibiotics has led to the generation of drug-resistant bacterial strains [77]. Additionally, research

and development efforts towards new antibiotics have been insufficient to keep pace with the rise

of antibiotic resistance [78]. As such, novel alternative approaches for the clinical management

of bacterial infections have become increasingly important. Along these lines, various

biomimetic prophylactics and therapeutics have demonstrated potential for overcoming the

challenges posed by antibiotic-resistant bacteria [49, 58, 79-82]. The use of BMVs in the design

of antibacterial vaccines represents a compelling approach. These naturally derived membrane

vesicles serve as an excellent training tool for the immune system due to their nanoscale size,

wide range of bacterial antigens, and inherent adjuvanting properties. They can also be

genetically engineered or paired with other nanomaterials to further enhance their utility.

4.1.1 Gram-negative bacteria

Gram-negative bacteria have a thin peptidoglycan cell wall and the presence of a

secondary exterior membrane from which outer membrane vesicles (OMVs) are naturally

released [20]. OMVs display antigens specific to their source bacteria along with PAMPs such as

LPS, which function as adjuvants for the induction of strong immune responses [83]. Efforts to

enhance the base characteristics of OMVs have also been undertaken through processes such as

genetic engineering and combination with nanoparticle-based platforms. Overall, OMVs have

demonstrated remarkable success as vaccines against a wide variety of bacteria. Most notably,

the clinically available Bexsero vaccine has been approved for use against N. meningitidis [84].

This vaccine is composed of meningococcal OMVs that serve as a source of porin A, along with

13
three recombinant antigen factors: H binding protein, neisserial adhesin A, and neisserial

heparin-binding antigen [85]. Interestingly, this same vaccine formulation has also demonstrated

the ability to induce protective antibody titers against the similar bacteria Neisseria gonorrhoeae,

which points to the potential versatility of multiantigen display using OMV-based vaccines.

OMVs isolated from multidrug-resistant A. baumannii strains have also shown the ability

to protect against the source pathogen in murine models [24, 86]. Improvements were achieved

through increases in specific antibody production that helped to reduce bacterial load. One study

immunized mice with ATCC 19606 OMVs and found near-perfect survival rates against the

source bacterial strain and two other independent A. baumannii strains that were otherwise over

80% lethal [86], once again emphasizing the potential of cross-protective immunity afforded by

OMV vaccines. A different study employing OMVs derived from an A. baumannii strain

afflicting intensive care unit patients also reported survival advantages [24]. In both sepsis and

pneumonia models of infection, vaccinated mice showed enhanced survival and reduced

bacterial loads. It was demonstrated that this OMV vaccine facilitated the production of antisera

with opsonophagocytic properties. Similar OMV-based vaccine approaches have also shown

success against other Gram-negative bacteria such as B. pertussis, Burkholderia mallei,

Burkholderia pseudomallei, Edwardsiella tarda, E. coli, Klebsiella pneumoniae, P. aeruginosa,

and S. enterica, among others [25, 29, 87-94].

Genetically engineering source bacteria is an approach that has been used to improve

upon the inherent benefits offered by OMVs. Bacteria can be manipulated to increase their OMV

production, decrease their toxicity, or modulate their antigen expression profile. For example,

Salmonella Paratyphi were transformed with a plasmid encoding the Salmonella Typhi-specific

Vi antigen to induce its presentation on the outer membrane of the bacteria [95]. This caused

14
OMVs shed by the bacteria to display both the Vi antigen from Salmonella Typhi and the O:2

antigen from Salmonella Paratyphi. Mice immunized with the bivalent OMVs developed

significant antibody titers specific against both bacteria, highlighting the potential of genetic

engineering approaches for enhancing the utility of OMV-based vaccination platforms. In

another example, E. coli OMVs were genetically modified to display Omp22, which is an outer

membrane protein from A. baumannii bacteria [96]. Surface presentation of the Omp22 antigen

was achieved by fusing it to ClyA found on E. coli OMVs. Active vaccination with the

engineered OMVs significantly reduced bacterial load and decreased serum cytokine levels in

mice challenged with A. baumannii relative to various controls. Furthermore, mice immunized

with Omp22-expressing OMVs achieved a 100% survival rate when challenged with an

otherwise lethal dose A. baumannii, whereas mice receiving wild-type OMVs only had a 63.6%

survival rate. Genetically engineering antigens onto BMVs has shown efficacy against a number

of other bacterial infections, including Orientia tsutsugamushi, Chlamydia muridarum, and

Francisella tularensis [97-99].

Besides naturally secreted OMVs, other Gram-negative bacterial membrane platforms

have been established for vaccine applications. In an example, nitrogen cavitation was employed

to produce double-layered membrane vesicles (DMVs) comprised of both the inner and outer

membranes of P. aeruginosa (Fig. 4) [100]. At 250 nm in size, these DMVs were shown to be

larger on average than OMVs. When administered in vivo, the DMVs were capable of generating

a multifaceted immune response as demonstrated by increases in antigen-specific IgG titers, the

maturation markers of DCs, and interleukin (IL)-2 levels from splenic T cells. Importantly, the

DMVs significantly outperformed an OMV control by improving survival after a lethal P.

aeruginosa challenge. Along similar lines, another work employed a process in which K.

15
pneumoniae were passed through a high-pressure homogenizer, causing them to break apart and

self-assemble into bacterial biomimetic vesicles (BBVs) [101]. This process allowed for facile

high-yield production, and the BBVs contained little intracellular protein or nucleic acid

contamination. In a murine model, the BBVs promoted a potent immune response that protected

against lethal K. pneumoniae challenges.

The integration of BMVs with other nanomaterials can help to enhance vaccine efficacy

by combining the benefits inherent to each component [47, 102]. This strategy has been utilized

by coating 30-nm gold nanoparticles with OMVs to produce a nanovaccine against E. coli

infection (Fig. 5) [57]. The gold nanoparticle cores provided a stable substrate for the OMVs,

thus reducing the average size while improving uniformity. This enabled the formulation to

readily drain into the lymph nodes following subcutaneous injection in vivo, leading to improved

DC maturation as confirmed by significant increases in surface maturation markers such as

CD40, CD80, and CD86. Vaccination with the OMV membrane-coated nanoparticles also

induced high avidity anti-E. coli IgG titers while promoting a Th1/Th17-biased T cell response.

A similar system used bovine serum albumin nanoparticles coated with OMVs from

carbapenem-resistant K. pneumoniae [103]. The nanovaccine induced strong DC maturation and

specific antibody titer production, thus protecting vaccinated mice from a lethal bacterial

challenge. Other enhancement strategies have involved the combination of OMVs with chitosan

for mucus adhesion [104] or zein nanoparticles coated with a Gantrez AN-119–mannosamine

conjugate to increase mucus permeation [105].

4.1.2 Gram-positive bacteria

Gram-positive bacteria possess a singular inner cytoplasmic membrane surrounded by a

thick peptidoglycan cell wall [106]. While these bacteria are capable of releasing extracellular

16
vesicles (EVs), the process is much less understood compared to the formation of OMVs in

Gram-negative bacteria. Regardless, efforts have been undertaken to study and develop Gram-

positive EVs for vaccine applications. It was discovered that phenol-soluble modulins and

autolysins were key factors in the release of S. aureus EVs by weakening the cytoplasmic

membrane and cell wall, respectively [107], and this information could be leveraged in the future

implementation of BMV-based vaccination strategies against Gram-positive bacteria. This study

also assessed the vaccination efficacy of EVs isolated from a mutant S. aureus strain that

expressed detoxified cytolysins. The formulation elicited the safe production of toxin-specific

antibody titers that provided a significant survival advantage to mice challenged with different S.

aureus strains.

Due to the complex and poorly understood nature of Gram-positive EV production, many

BMV-based vaccination efforts have instead opted to leverage genetically engineered OMVs

from Gram-negative bacteria. For example, E. coli cells were modified to express heterologous

group A Streptococcus (GAS) antigens in their periplasmic space for inclusion in the lumen of

OMVs [108]. The natural immunogenicity of the E. coli OMVs promoted the production of GAS

antigen-specific antibody titers that helped improve the survival rate of immunized mice when

exposed to GAS. Another genetic engineering strategy created detoxified E. coli OMVs

displaying poly-N-acetyl-D-glucosamine (PNAG), which is a commonly expressed antigen on

different pathogens [109]. For this study, the researchers were able to engineer PNAG-

expressing OMVs with variable degrees of acetylation, and it was determined that deacetylated

PNAG evoked the strongest specific antibody titers. This resulted in better survival rates for

immunized mice challenged with either Gram-positive S. aureus or Gram-negative F. tularensis.

Overall, the study emphasized the potential that genetic engineering holds for the development of

17
broadly protective vaccines. Genetically engineered BMVs have also shown success against

other Gram-positive bacteria such as Streptococcus pneumoniae [42, 110].

Biomaterial-based enhancement strategies are also being utilized in the development of

BMV vaccines against Gram-positive bacteria. For instance, mesoporous silica nanoparticles

were loaded with indocyanine green and then coated with S. aureus EVs [111]. Laser irradiation

of DCs after uptake of the nanoformulation resulted in elevated local temperature that facilitated

endosomal escape, thus increasing the production of reactive oxygen species and enhancing

proteasome activation. This allowed for the efficient downstream activation of both CD4+ and

CD8+ T cell immune responses. In an in vivo efficacy study, immunized mice exhibited

decreased lesion formulation and lower bacterial loads when challenged with S. aureus.

4.2 Viral infections

Viruses can be highly contagious and have been responsible for some of the most

pressing public health crises in recent history. While there are a limited number of antiviral

therapeutics available, vaccination has been a highly effective strategy for the management of

viral infections. The smallpox vaccine, which employed a live-attenuated version of the virus,

was developed by Dr. Edward Jenner in the 18th century [112]. Viral prophylactics have since

been developed using a variety of strategies that include inactivated vaccines, subunit vaccines,

and, most recently, mRNA vaccines [113-115]. While many advances have been made, there is

still a strong need for viral vaccines that are more effective, safer, and readily adaptable to

emerging pandemics. BMVs are suitable for viral vaccine development due to their inherent

immunogenicity and ability to display non-native antigens. Bacteria-derived formulations have

been developed to combat a wide range of viruses, including those responsible for tropical

diseases, sexually transmitted infections, and respiratory ailments [116-119].

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4.2.1 Influenza

Various strains of influenza place a large burden on the healthcare system annually. The

most common influenza vaccines include intramuscularly administered quadrivalent inactivated

virus mixtures (Fluzone, Fluarix, and FluLaval) and a quadrivalent live-attenuated virus

delivered by a nasal spray (FluMist) [120]. While they can be highly effective, these vaccines

oftentimes fail to predict the most prevalent annual strains and may only confer partial immunity

[121]. Whole bacterial vectors, such as non-pathogenic Gram-positive lactic acid bacteria, have

been engineered to express influenza antigens alongside natural adjuvants to induce antigen-

specific immunity [122]. In addition to these whole cell platforms, bacterial OMVs have shown

strong potential to carry influenza antigens for effective vaccination [14]. OMVs are often first

modified to reduce their toxicity, which is largely mediated by the presence of LPS on their

surface [123]. Because bacterial OMVs are already strongly immunogenic, there is oftentimes no

need to further engineer them to include an adjuvant. In one example, E. coli OMVs co-

expressing a stable chimeric fusion protein of H1N1 and Middle East respiratory syndrome

coronavirus antigens were produced and administered intramuscularly to mice [124]. The

vaccinated mice generated antibody titers against both heterogenous antigens and demonstrated

improved survival when challenged with H1N1 influenza. In addition to being engineered to

express viral antigens, OMVs can also be used directly as co-administered adjuvants to enhance

cellular immunity [125]. As compared to the widely used alum, endotoxin-attenuated E. coli

OMVs promoted equivalent antiviral titer generation when used as an adjuvant; additionally, the

bacterial vesicles induced higher IgG2c:IgG1 ratios and IFN-γ production.

While there has been increased interest in bacteria-mediated influenza vaccination in the

past decade, the majority of ongoing and completed clinical trials in this field have focused on

19
the use of probiotic administration as an adjuvant for already approved influenza vaccines, or

they have failed to elicit satisfactory influenza-specific antibody generation in humans [126,

127]. Continued work to improve the safety profile and immunogenicity of bacteria-based

influenza vaccines, along with an increased focus on the use of BMVs as the vector or adjuvant,

may enable the successful development of a clinically viable formulation in the future.

4.2.2 Coronaviruses

The ongoing coronavirus disease 2019 (COVID-19) pandemic, having infected over 286

million people and killed over 5.4 million as of December 2021, has highlighted the necessity of

rapid vaccine development in the face of fast-spreading disease. Severe acute respiratory

syndrome coronavirus 2 (SARS-CoV-2) utilizes the receptor binding domain (RBD) of its spike

protein to enter host cells [128]. Because of the importance of this process in the propagation of

SARS-CoV-2, the spike protein has become the primary target for COVID-19 vaccines, yielding

multiple formulations that have received an Emergency Use Authorization in the United States

[129]. Vaccination with the protein alone yields little to no immune protection, and thus the

antigen must be delivered in an immunostimulatory format. Several works have explored the use

of whole bacteria-based vectors to express and deliver SARS-CoV-2 antigens to promote strong

protective immunity against COVID-19 [130, 131].

Due to their ease of engineering and their inherent adjuvating nature, BMVs have also

been utilized in the development of novel COVID-19 vaccine formulations. Along these lines,

the co-administration of SARS-CoV-2 antigens and immunostimulatory BMVs as adjuvants has

shown promise. In one instance, recombinant RBD was co-administered with N. meningitidis

OMVs [132]. Vaccination with the mixture elicited anti-RBD IgG and IgA titers as well as

production of IFN-γ and IL-17. Bacterial OMVs can also be engineered to directly express RBD

20
on their surface [133-135]. As an example, ClyA was fused to RBD and expressed in E. coli,

resulting in display of the viral antigen on the surface of the OMVs. BMV yield is often a

translational challenge, and a recent work addressed this by subjecting ClyA–RBD-expressing

bacteria to high-pressure homogenization, which increased RBD loading by 107-fold (Fig. 6)

[135]. Vaccination of mice with the RBD-expressing BMVs generated antigen-specific IgGs that

were capable of blocking the interaction between the SARS-CoV-2 spike protein and its

receptor, angiotensin converting enzyme 2 (ACE2).

4.2.3 Human immunodeficiency virus

Despite over 40 years of research, an effective vaccine for human immunodeficiency

virus (HIV) remains elusive [136]. As of 2020, there are 37.7 million cases of HIV worldwide

with an estimated 1.5 million new cases yearly [137]. Current approaches to HIV vaccination

have exhibited adverse effects, low targeting capabilities, and poor bioavailability [138]. As

such, development of a safe and effective vaccine against the virus is of critical importance.

A novel DNA vaccine platform comprised of BMVs from Salmonella Typhi Ty21a was

used to deliver an HIV-1 glycoprotein, gp140, as a vaccine against HIV-1 [118]. In this platform,

BMVs were derived from lysed Salmonella Typhi, and DNA encoding for gp140 was loaded

through chemical destabilization of the BMV membrane with sodium acetate. Mice vaccinated

with the DNA-loaded BMVs showed significantly higher mucosal antibody responses as well as

increased IL-10 production. Another platform leveraged bacterium-like particles (BLPs) based on

Lactococcus lactis to generate mucosal and humoral immunity against HIV-1 [139]. In the study,

gp120 was conjugated to a peptidoglycan-binding tag capable of attaching to the L. lactis cell

wall. With intranasal vaccination, the gp120-functionalized BLPs successfully induced mucosal

immunity in mice and produced neutralizing activity against HIV-1 pseudoviruses in guinea pigs.

21
3.2.3 Hepatitis viruses

The hepatitis viruses are a set of five viruses that cause liver inflammation [140].

Currently, the most medically relevant are hepatitis A, B, and C, with the hepatitis B virus

(HBV) causing the majority of hepatitis-related deaths. While there is a safe and effective

vaccine to protect against HBV, a hepatitis C virus (HCV) vaccine has yet to be developed [141].

While direct-acting antiviral treatments are effective against HCV infection, prevention remains

a primary goal. Along these lines, L. lactis was engineered to produce polyhydroxybutyrate

inclusions displaying the hepatitis C virus core antigen [142]. Vaccination of mice with the

antigen-presenting particles resulted in increased production of IFN-γ, IL-17A, tumor necrosis

factor α, and IL-6, as well as low levels of IgG2c antibodies. With administration alongside the

adjuvant Emulsigen, a strong IgG1 antibody and cytokine response was generated. Another

vaccination approach against HCV leveraged a fusion protein encoding for a truncated portion of

the core antigen fused to the nonstructural protein 3 of HCV [143]. The antigen was then

admixed with purified N. meningitidis BMVs and administered subcutaneously to mice. This

formulation was able to generate high levels of IgG antibodies, proinflammatory cytokines, and

granzyme B, indicating potential as an HCV vaccine candidate.

A BMV-based vaccine against HBV was developed by anchoring HBV core protein to

either the inner or outer membrane of E. coli [144]. The antigen was genetically fused with

OmpA protein as an anchor, enabling it to be translocated to the membrane layers. Compared

with purified core protein, the modified BMVs were able to induce higher levels of HBV-

specific IgG antibodies. A related study expressed the HBV core antigen in attenuated

Salmonella Typhimurium and Salmonella dublin bacteria strains [145]. These live recombinant

bacteria were able to generate high titers after a single oral immunization.

22
4.2.5 Human papillomavirus

Human papillomavirus (HPV) is a small non-enveloped DNA virus that represents the

main causative agent of cervical cancer in women. Fortunately, two prophylactic HPV vaccines,

Gardasil and Cervarix, have been approved for clinical use, providing protection against HPV

types 6, 11, 16, and 18, which together cause approximately 70% of cervical cancer. When

administered prophylactically, these vaccines have a near 100% protection rate [146]. While

extremely effective, HPV vaccines are expensive and require a cold chain, which negatively affect

their accessibility. In addition, the vaccines are developed against the L1 capsid protein, which,

although effective, is highly type-specific. In contrast, vaccination with the L2 capsid protein has

been shown to generate broad cross-neutralizing antibodies against HPV, although the antigen on

its own has low immunogenicity [147]. A common bacteria-mediated approach to developing HPV

vaccines has been the surface display of antigens such as L2 on orally administered bacteria. In

one instance, HPV-16 L2 protein was expressed on the surface of Lactobacillus casei and orally

administered to BALB/c mice periodically over the course of a month [148]. Mice vaccinated with

the engineered bacteria developed serum and mucosal IgG and IgA titers that protected them

against challenge with heterologous and homologous pseudoviruses. While BMV-based vaccines

for preventing HPV have not yet been reported, there is potential for future development along

these lines.

Although beneficial for HPV-naïve individuals, prophylactic vaccination strategies

cannot be applied to those who already infected. As such, therapeutic vaccines designed against

HPV-driven malignancies have also been of great interest to researchers. In these cases,

oncogenesis-related proteins, such as E6 and E7, serve as the main antigenic targets. Listeria

monocytogenes is a bacterium that has demonstrated suitability as a therapeutic HPV vaccine

23
vector, as it elicits durable CD4+ and CD8+ T cell responses [149]. Axalimogene filolisbac,

which consists of live-attenuated L. monocytogenes expressing E7 fused to listeriolysin O, is an

immunotherapy currently undergoing clinical trials [146]. Preliminary data show vaccine safety

and improvements in survival rate for those suffering from late-stage HPV-induced cancers. L.

lactis is another bacterial strain that has shown promise as a vector for delivering the E7 protein

in an immunostimulatory context [150, 151]. In terms of BMV-based solutions, the engineering

of E7 onto E. coli OMVs has been shown to elicit antigen-specific cellular immunity [119].

When administered therapeutically, the nanoformulation significantly suppressed tumor growth

in a TC-1 model.

4.3 Cancer

Beyond vaccines to prevent pathogenic infections, bacteria-derived vesicles have been

utilized for cancer immunotherapy. Owing to a wide abundance of PAMPs, bacteria are highly

immunogenic and can effectively recruit and activate immune cells at tumor sites [152-154].

However, the use of intact bacteria poses safety concerns [155], and thus an alternative approach

has been to employ bacterial ghosts that are void of intracellular contents [156, 157]. The

hollowed-out bacteria can be further loaded with adjuvants or antigenic payloads to enhance

efficacy and specificity. Compared with their free form counterparts, antigens loaded into

bacterial ghosts have demonstrated better retention after subcutaneous administration, increased

immune activation, and improved tumor growth suppression [158]. In addition to bacterial

ghosts, OMVs have also been used to overcome immunosuppressive tumor microenvironments

[159]. The small size of OMVs enables efficient lymphatic drainage upon subcutaneous injection

and enhanced localization to solid tumors through passive targeting effects when introduced

systemically [160]. As OMVs can naturally accumulate at tumor sites and utilize local cancer

24
cells as antigen sources in situ, they can be applied therapeutically against multiple types of

cancer. For example, administration of a low intravenous dose of E. coli OMVs resulted in

complete tumor eradication in CT26 and MC38 colorectal cancer, 4T1 metastatic breast cancer,

and B16BL6 metastatic melanoma models.

A major advantage of BMVs for cancer vaccine applications is the flexibility afforded by

their ease of genetic engineering. As an example, E. coli was genetically modified to release

OMVs in situ in response to the presence of arabinose [161]. The secreted OMVs contained

personalized tumor antigens and an Fc fragment for enhanced DC uptake and transcytosis across

the intestinal epithelium. Oral administration of the reprogrammed E. coli along with arabinose

effectively prevented lung metastasis in a B16-OVA model and controlled tumor growth in an

MC38 subcutaneous tumor model. In another instance, genetic engineering was employed to

express basic fibroblast growth factor (BFGF) on OMVs to elicit autoantibodies [162]. BFGF is

an angiogenic molecule with numerous pro-tumorigenic functions such as augmenting tumor cell

proliferation, promoting metastasis, inhibiting apoptosis, and enhancing immune suppression

[163, 164]. Immune-mediated removal of BFGF effectively disarmed tumors of a vital tool

utilized for their survival. Another strategy to engineer proteins onto OMVs has been through

fusion with ClyA, which was exemplified by the expression of programmed cell death protein 1

(PD-1) on OMVs to prevent T cell exhaustion [165]. OMVs have also been engineered such that

they can be rapidly modified after secretion in a modular manner [166]. In this case, ClyA was

fused with either SpyCatcher or SnoopCatcher, thus enabling rapid capture of tumor neoantigens

with the corresponding SpyTag or SnoopTag modifications, respectively. Utilizing this plug and

display system, potent antitumor immunity was achieved in a B16-F10 metastasis model and an

MC38 colorectal cancer model.

25
 Cancer immunotherapy with OMVs has been combined with other types of therapeutics

to synergistically amplify antitumor efficacy. As OMVs are nanovesicles that can be localized to

tumor sites, chemotherapeutics such as doxorubicin and paclitaxel have been encapsulated inside

[152, 167]. Compared to OMVs alone, this chemoimmunotherapy approach has resulted in

significantly better attenuation of tumor growth. In another example, OMVs derived from

attenuated Salmonella was functionalized with an RGD tumor-targeting peptide and coated onto

polymeric micelles loaded with tegafur, a prodrug of fluorouracil that not only induces tumor

apoptosis, but also sensitizes cancer cells to cytotoxic T lymphocytes [168]. Another common

combinatorial approach is photoimmunotherapy, which leverages photosensitizers such as

indocyanine green [169]. After accumulation in tumors, laser ablation releases numerous cancer

antigens for immune priming. In the absence of photothermal agents, Salmonella Typhimurium

and its secreted OMVs were found to naturally sensitize tumors for photothermal therapy

through tumor darkening caused by thrombosis and the extravasation of red blood cells [170,

171]. Purely external approaches to tumor destruction such as radiation therapy can also be used

to augment the effectiveness of OMVs [172]. To further improve treatment outcomes in this type

of scenario, OMVs were modified with maleimide, allowing them to capture tumor antigens

released after radiotherapy [173]. Moreover, the modified OMVs were wrapped onto a polymeric

core designed for endosomal escape and loaded with CpG for additional immune stimulation.

Using this platform, significant tumor eradication was achieved in B78 melanoma and NXS2

neuroblastoma models. A unique combinational immunotherapy was achieved with OMV-loaded

micromotors (Fig. 7) [174]. The micromotor formulation was injected directly into tumors,

resulting in considerable physical disruption and damage. Necrotic tumor cells were

subsequently processed by infiltrating immune cells, leading to systemic antitumor immunity.

26
Compared to a non-propelling micromotor control, treatment with the active OMV-loaded

micromotors in MC38, CT26, and B16-F10 tumor models resulted in considerable tumor

clearance at the primary site and improved control of distal tumor growth.

Antigen-rich cancer cell membrane vesicles and exosomes have been incorporated with

bacteria-derived cancer vaccines to increase their specificity.[175] Along these lines, the co-

administration of B16-F10 exosomes and synthetic E. coli vesicles was shown to control tumor

growth and metastasis, in addition to sensitizing tumors to anti-PD-1 therapy [176]. The

synthetic bacterial vesicles were purified from intact bacteria through treatment with lysozyme

and high pH to eliminate periplasmic and cytosolic proteins, followed by dissolution in an ionic

detergent to solubilize the inner membrane. This ultimately resulted in the generation of vesicles

with an improved safety profile that maintained strong adjuvanticity. Beyond co-administration,

other strategies have adopted hybrid vesicles synthesized by fusing cancer cell membrane with

bacteria-derived membrane [177]. Compared with the simple mixture of pure cancer cell

membrane vesicles and bacterial vesicles, a fusion membrane platform guarantees colocalization

of the antigens and adjuvant, which can significantly enhance immune activation and treatment

outcomes [178]. Using this approach, mice that were vaccinated with nanoparticles coated with a

fusion membrane composed of melanoma membrane vesicles and attenuated Salmonella OMVs

were fully protected from tumor challenge [179]. In a therapeutic setting, efficacy was further

amplified when combined with photothermal therapy [179, 180].

5. Summary and outlook

BMVs contain a number of features that make them attractive for vaccine development.

This includes their ability to display proteins from different sources, natural presence of PAMPs

27
for eliciting strong immune responses, nanoscale size for efficient antigen delivery and

processing, and flexibility to be further engineered as necessary. Leveraging these properties, a

wide range of BMV-based nanovaccines have been developed against pathogens such as bacteria

and viruses, as well as different types of cancer. While BMV nanovaccines have demonstrated

considerable promise, there are still many challenges that these platforms face in terms of

clinical translation. First, because they must be harvested from living bacteria, there is inherent

batch-to-batch variability in size, antigen density, and overall composition that must be

accounted for during large-scale manufacturing. Strategies to lower variance include the

standardization of BMV harvesting procedures and the use of low passage number bacterial

stocks. Additionally, the coating of BMVs onto nanoparticle substrates using methods such as

physical extrusion can help to tighten the size distribution of the final formulation while

maintaining protein integrity. Stabilization using nanoparticle cores also helps to facilitate

improved uptake by APCs, thus ensuring proper antigen processing and presentation. Another

strategy for improving stability could involve the production of multi-layered crosslinked

membrane vesicles.

While the immunostimulatory properties of PAMPs are attractive for vaccine

applications, this must be balanced with potential safety concerns associated with the

introduction of endotoxins such as LPS into human patients. Research is needed to develop more

effective isolation and purification techniques that can more precisely separate harmful bacterial

components from BMVs. Novel genetic engineering strategies for reducing the expression of

toxic molecules are highly promising, and they could ultimately lead to the production of BMVs

that are inherently safe without the need for sophisticated processing. Furthermore, repeated

vaccinations with BMVs may induce an immune response towards the carrier itself, which could

28
create off-target immune responses that attenuate immunity against the intended target antigen.

This issue may be addressed by boosting antigen density through various engineering

approaches. Nonessential proteins that are particularly immunogenic could also be

downregulated or knocked out. As BMV platforms become more refined, an increased emphasis

will be placed on the rational design of multi-antigenic formulations. This can streamline vaccine

production and overcome the need for mixing multiple antigens together, which is currently

required for the clinically approved Bexsero formulation.

While there is still significant room for improvement, great strides have already been

made in the development of BMV platforms for the clinical management of bacterial infections

and cancer [181, 182]. Continued research on BMVs will undoubtedly yield new and innovative

solutions that will address current challenges and enable mainstream adoption in the clinic.

Acknowledgement

This work is supported by the Defense Threat Reduction Agency Joint Science and

Technology Office for Chemical and Biological Defense under Grant Number HDTRA1‐18‐1‐

0014.

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Table and table captions

Class of PRR PRR PAMP Signaling pathway

TLR1, Triacyl lipopeptides, diacyl
NF-κB signaling
TLR2 lipopeptides
TLR2, Lipoteichoic acid, peptidoglycans,
NF-κB signaling
TLR6 lipoproteins, pore proteins
Toll-like receptor NF-κB signaling; TRIF-
TLR4 Lipopolysaccharides
(TLR) dependent pathway
TLR5 Flagellin NF-κB signaling
TLR6 Lipoteichoic acid, peptidoglycans NF-κB signaling
TLR9 Non-methylated CpG DNA NF-κB signaling
NOD1 Diaminopimelic acid NF-κB signaling; MAPK
Nucleotide-binding signaling; IRF signaling
NOD2 Muramyl dipeptide
oligomerization domain-like
NLRP1 Muramyl dipeptide NF-κB signaling; NLRP
receptor (NLR)
NLRP3 RNA, DNA, muramyl dipeptide inflammasome
Stimulator of interferon STING Cytosolic DNA NF-κB signaling; IRF3
genes (STING) signaling

Table 1. Bacterial PRRs, PAMPs, and their corresponding signaling pathways. PRRs found on

host cells can be activated by various bacterial PAMPs, which in turn activate inflammatory

pathways to generate an immune response [62, 63].

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Figures and figure captions

Fig. 1. Bacterial membrane vesicles (BMVs) for vaccine applications. BMVs are derived from

bacteria and can be leveraged in the design of effective nanovaccines through common

engineering approaches including genetic surface modification, cargo loading, and nanoparticle

coating. The resulting vaccine formulations have shown promise against a wide range of

diseases, including bacterial infections, viral infections, and cancers.

54
Fig. 2. BMVs for biomedical applications. (A) BMVs can be isolated and purified from bacterial

culture supernatant using filtration followed by density gradient centrifugation. Adapted with

permission [29]. Copyright 2021, Springer Nature. (B) Electroporation can be used to generate

pores on BMVs to facilitate cargo loading. Adapted with permission [32]. Copyright 2019,

Springer Nature. (C) A SpyTag/SpyCatcher system can be used to covalently link payloads to

the surface of BMVs. Adapted with permission [46]. Copyright 2015, American Chemical

Society. (D) BMVs can be coated onto the surface of nanoparticle cores, which aids in

stabilization and can provide additional functionality. Adapted with permission [58]. Copyright

2019, Wiley-VCH.

55
Fig. 3. Activation of the immune system by BMVs. (A) Pathogen-associated molecular patterns

(PAMPs) from BMVs induce maturation of antigen-presenting cells such as dendritic cells (DCs)

via interaction with pattern recognition receptors (PRRs). Mature DCs process and present

exogenous antigens via major histocompatibility complexes (MHCs) alongside costimulatory

markers such as CD80 or CD86, promoting the activation of naïve T cells. (B) Differentiated

helper T cells interact with B cells to stimulate the production of antigen-specific protective

antibodies. (C) Differentiated cytotoxic T cells are capable of directly killing cells expressing the

target antigen.

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Fig. 4. P. aeruginosa double-layered membrane vesicle (DMV) vaccine. (A) DMVs are

generated by subjecting P. aeruginosa to nitrogen cavitation, followed by differential

centrifugation. (B) DMVs outperform an OMV-based formulation in the generation of protein-

specific IgG titers. (C) Mice immunized with DMVs exhibit an improved survival rate compared

with OMV-immunized mice when challenged with a lethal dose of P. aeruginosa. Adapted with

permission [100]. Copyright 2018, Elsevier.

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Fig. 5. E. coli OMV-coated gold nanoparticle vaccine. (A) OMVs are isolated from E. coli and

coated onto a gold nanoparticle core, and the resulting formulation can be used as a vaccine to

generate a protective immune response. (B) The bacterial membrane-coated gold nanoparticles

(BM-AuNPs) outperform free OMVs in eliciting anti-E. coli IgG titers in vivo. (C) BM-AuNPs

elicit increased production of proinflammatory cytokines and promote Th1/Th17-biased

immunity. Adapted with permission [57]. Copyright 2015, American Chemical Society.

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Fig. 6. Bacterial biomimetic vesicles (BBV) for COVID-19 vaccination. (A) E. coli expressing a

ClyA–RBD fusion protein are subjected to high pressure homogenization to yield ring-like RBD-

expressing BBV (RBD-BBV). (B) IgG responses are significantly increased in mice immunized

with RBD-BBV versus various controls. (C) Antibodies generated by RBD-BBV vaccination are

able to lower the affinity between the S1 binding domain of the SARS-CoV-2 spike protein and

its receptor, ACE2. Adapted with permission [135]. Copyright 2021, American Chemical

Society.

59
Fig. 7. OMV-loaded micromotors (Motor-OMV) for combination cancer immunotherapy. (A)

OMVs are purified from E. coli and loaded onto a self-propelling micromotor. When

administered intratumorally, the micromotors induce physical destruction, which, combined with

the immunostimulatory effect of the OMVs, elicits a systemic antitumor response. (B) After

intratumoral administration, the Motor-OMV formulation induces significant local disruption of

the tumor tissue. (C) Compared with a static OMV-loaded microparticle (MP-OMV) control,

Motor-OMV better control tumor growth in MC38 (top), CT26 (middle), and B16-F10 (bottom)

models. Adapted with permission [174]. Copyright 2021, Wiley-VCH.

60