R E S E A R C H A R T I C L E

Immunogenicityandprotective e⁄cacyofVibrio cholerae outermembranevesicles in rabbitmodelNivedita Roy1, Soumik Barman1, Amit Ghosh2, Amit Pal2, Krishnendu Chakraborty3, Santa Sabuj Das3,Dhira Rani Saha4, Shinji Yamasaki5 & Hemanta Koley1

1Division of Bacteriology, National Institute of Cholera and Enteric Diseases, Kolkata, India; 2Division of Pathophysiology, National Institute of Cholera

and Enteric Diseases, Kolkata, India; 3Division of Clinical Medicine, National Institute of Cholera and Enteric Diseases, Kolkata, India; 4Division of

Electron Microscopy, National Institute of Cholera and Enteric Diseases, Kolkata, India; and 5Graduate School of Life and Environmental Sciences, Osaka

Prefecture University, Osaka, Japan

Correspondence: Hemanta Koley, Division

of Bacteriology, National Institute of Cholera

and Enteric Diseases, P – 33 CIT Road, Scheme

–- XM, Beliaghata, Kolkata 700010, India.

Tel.: 191 33 2353 7470, Ext. 3020; fax: 191

33 2370 5066; e-mail:

hemantakoley@hotmail.com

Received 25 September 2009; revised 17 April

2010; accepted 26 April 2010.

Final version published online 7 June 2010.

DOI:10.1111/j.1574-695X.2010.00692.x

Editor: Patrik Bavoil

Keywords

outer membrane vesicles (OMVs); Vibrio

cholerae ; oral immunization; colonization;

rabbit model; RITARD model.

Abstract

We show here that oral immunization with purified outer membrane vesicles

(OMVs) of Vibrio cholerae induces a prolonged high rise in the protective antibody

titre. Rabbit immune sera were vibriocidal against the homologous and against

several heterologous V. cholerae strains in vitro. In addition, OMV immunization

conferred significant protective immunity against subsequent bacterial challenges.

Thirty OMV-immunized rabbits were challenged with different V. cholerae strains;

each challenged group contained five immunized and three unimmunized

animals. All the immunized rabbits survived bacterial challenges and were healthy

after 24 h, except the two from each group that received the SG24 and SG06 strains,

respectively, which developed watery diarrhoea. In contrast, all the unimmunized

animals developed cholera-like symptoms, with a death toll of eight within 24 h of

challenge. This is the first report of the induction of protective immunity by

V. cholerae OMVs in a rabbit model (removable intestinal tie-adult rabbit

diarrhoea) that mimics the human disease. Finally, OMVs were found to be

significantly less reactogenic than the live and the heat-killed bacteria. Our studies

show that oral immunization with OMVs of V. cholerae may induce long-term

immunity and may be useful as a ‘nonliving’ vaccine candidate for the future.

Introduction

Enteric bacterial infection is the major cause of diarrhoea,

particularly in the developing world (Mathan, 1998). Chil-

dren and adults living in the less developed world experience

frequent diarrhoeal episodes, which may lead to loss of

working hours, costly hospitalization and sometimes death

(Monroe et al., 1991). Furthermore, travellers to developing

countries are exposed to the high risk of infectious diar-

rhoea, resulting in discomfort and inconvenience. Although

the mortality from diarrhoeal diseases has been significantly

reduced for some time, primarily due to better quality of

drinking water, improved sanitation and the availability of

more effective antimicrobial agents, the disease burden and

as a consequence the morbidity and economic fallout still

remain substantial. Vibrio sp. constitutes one of the leading

causes of acute watery diarrhoea. Cholera, sometimes

known as Asiatic or epidemic cholera, is an infectious

gastroenteritis caused by enterotoxin-producing strains of

the bacteria Vibrio cholerae that leads to epidemic cases of

gastroenteritis and sporadic cases of septicaemia. However,

epidemic V. cholerae isolates are represented by only two

serogroups, namely the O1 and O139, of a total of 209

different serogroups identified so far on the basis of the

somatic O-antigen (Chatterjee & Chaudhuri, 2003).

Non-O1 and non-O139 V. cholerae species have only been

associated with sporadic cases of diarrhoea (Faruque et al.,

1998).

Although cholera can be life threatening, it can be

prevented by proper sanitation and supply of bacteriologi-

cally pure drinking water. However, the economic cost of

infrastructural development to ensure water supply and

sanitation facilities to every citizen is huge and has not yet

been achieved in India, where cholera is endemic. A cheaper

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd.

IMM

UN

OLO

GY

& M

EDIC

AL

MIC

ROBI

OLO

GY

alternative for individual and community-level protection

against cholera might be provided by an economical and

effective vaccine that would mitigate the spread of the

disease. Parenteral cholera vaccines are no longer in use,

but both live and killed whole cell vaccines are currently

available on the market and have been found to be safe,

immunogenic and effective in defined populations. These

include killed whole cell-recombinant cholera toxin B sub-

unit vaccine (WC/rCTB; Dukorals, Crucel/SBL, Sweden)

(Richie et al., 2000), killed whole vaccine (the Vietnamese

vaccine; ORC-Vaxs, VaBiotech) and modified Vietnamese

vaccine (polyvalent killed whole cell; Shancols, Shanta

Biotech, India). The only licensed live oral vaccine,

CVD103-HgR (Orochols, Crucel/Berna Biotech, Switzer-

land), has not been produced since 2004. A lack of efficacy in

certain populations and reactogenicity have remained the

major bottlenecks preventing the wider use of the available

cholera vaccines, particularly in developing countries. Sev-

eral research groups have recently shifted their focus to the

development of subunit vaccines. However, results from

animal experiments suggest that such vaccines may only be

effective when administered with an adjuvant, and serious

concerns have been raised that nonliving protein material

delivered onto mucosal surfaces may induce a state of

tolerance. Even cholera toxin or the B subunit, the two

potent mucosal adjuvants for the induction of antibody

responses, may actually be tolerogenic when it comes to

T-cell-dependent immunity. There exists a need to develop a

new vaccine that can offer long-term protection against

infections with a wide range of V. cholerae serogroups and

that at the same time causes fewer adverse effects.

A wide variety of Gram-negative bacteria, such as Escher-

ichia coli, Neisseria meningitidis, Pseudomonas aeruginosa,

Shigella flexneri, Helicobacter pylori and V. cholerae constitu-

tively secrete outer membrane vesicles (OMVs) during their

growth in vitro (Chatterjee & Das, 1967; Devoe & Gilchrist,

1973; Hoekstra et al., 1976; Kondo et al., 1993; Fiocca et al.,

1999; Kadurugamuwa & Beveridge, 1999). Recent studies

with mice have shown promise for the use of OMVs to

provide protection against infections with N. meningitidis

(Gonzaleza et al., 2006), Salmonella enterica serovar Typhi-

murium (Alaniz et al., 2007), H. pylori (Keenan et al., 2000)

and V. cholerae (Schild et al., 2008). OMVs are spherical,

bilayered lipoproteins with an average diameter of

20–200 nm. They are composed of outer membrane pro-

teins, lipopolysaccharide, outer membrane lipids, periplas-

mic proteins, cytoplasmic proteins, DNA, RNA and other

factors associated with virulence (Horstman & Kuehn, 2000;

Wai et al., 2003; Kuehn & Kesty, 2005; Bauman & Kuehn,

2006; Nevot et al., 2006; Lee et al., 2008).

Studies of OMVs from diverse bacterial strains suggest a

role in the delivery of toxins to host cells, the transfer of

proteins and genetic material between bacterial cells, cell-to-

cell signals and the elimination of competing organisms

(Kuehn & Kesty, 2005; Mashburn-Warren & Whiteley,

2006). Because OMVs are essential to bacterial survival in

the host and pathogenesis, modulation of vesicle formation

and their functions may be a useful objective in relation to

the development of antibiotics as well as vaccines (Henry

et al., 2004; Lee et al., 2008). As outer membrane proteins

and lipopolysaccharide of OMVs may induce host immune

response, detergent-extracted OMVs derived from patho-

genic strains, including N. meningitides, are now undergoing

clinical trials (Fredriksen et al., 1991; de Moraes et al., 1992;

Edwards & Apicella, 2004; Davidsen & Tonjum, 2006;

Gorringe et al., 2009).

A vaccine developed from OMVs of group B meningo-

cocci suspended in saline has been proved to be strongly

immunogenic in mice and humans when administered

intranasally as drops or spray, even without the use of

mucosal adjuvants (Dalseg et al., 1999; Siadat et al., 2007;

Gorringe et al., 2009). Recently, Schild et al. (2009) reported

that V. cholerae OMVs also possess immunogenicity, and

mucosal immunization of mice with these OMVs induces

long-term protective immunity against this gastrointestinal

pathogen in the litters.

In the current study, we thoroughly investigated the

safety, immunogenicity and protective efficacy of OMVs

derived from V. cholerae and their potential use as an orally

administered candidate vaccine.

Materials and methods

Animals

New Zealand White rabbits of either sex weighing 2.0–2.5 kg

were used for oral immunization with OMVs and challenge

experiments in the removable intestinal tie-adult rabbit

diarrhoea (RITARD) model. All rabbits were collected from

the animal resource department of the National Institute of

Cholera and Enteric Diseases, Kolkata. They were individu-

ally caged and maintained at 24 1C with 75% humidity and

fed sterile food and water. Gut sterilization of the animals

was performed by treatment with a course of metronidazole

(125 mg per rabbit day�1) and sulphaquinoxaline sodium

(464 mg per rabbit day�1) for 5 days, and the course was

repeated three times at 2-day intervals to remove giardia and

coccidia from the intestine (Albert et al., 1991; Koley et al.,

1995). Approval was received from the institutional ethical

committee before the animal experiments were performed.

Bacterial strains

OMV antigen was prepared from the reference strain

N16961 (El-tor Inaba). The other V. cholerae strains that

were selected for the heterologous challenge experiments

were O395 (Classical Ogawa), 569B (Classical Inaba), VC20

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd.

19Vibrio cholerae OMVs vaccine candidate

(El-tor Ogawa), SG24 (O139) and SGO6 (O45, non-O1,

non-O139). All strains were stored in 15% glycerol with

brain heart infusion broth (BHIB, Difco) at � 70 1C.

OMV preparation

OMVs were isolated from the reference V. cholerae strain

N16961 following the method of Balsalobre et al. (2006).

Briefly, 1 L of Luria–Bertani broth (LB, Difco) was inocu-

lated with 10 mL of the stationary phase culture of N16961

strain and grown for 8 h to the late exponential phase.

Bacteria were removed by centrifugation (4500 g, 15 min,

4 1C), and the supernatant was filtered by passing it con-

secutively through 0.45-mm and 0.22-mm pore size filters,

respectively. To confirm the absence of viable bacteria, 1 mL

of the filtrate was plated on an LB agar plate, which was

incubated overnight at 37 1C. Protease inhibitors [Complete

EDTA-free protease inhibitor cocktail (Roche), 1 tablet per

1 L of filtrate] were added to the filtrate to prevent protein

degradation. OMVs were subsequently purified from the

filtrate by ultracentrifugation (4 h, 140 000 g, 4 1C) using a

Beckman SW32Ti rotor, washed once with phosphate-

buffered saline (PBS; pH 7.4) and finally resuspended in

625mL of PBS. Protein concentration was determined by the

Modified Lowry Protein Assay Kit (Pierce) and adjusted to

2.5mg mL�1 using PBS. Purified OMVs were stored at

� 80 1C until use.

Electron microscopy

A 5-mL aliquot of OMVs was deposited on the carbon-

coated grid, allowed to settle for approximately 2 min and

washed with Tris buffer solution. Excess fluid was blotted

and covered with a small drop of 2% aqueous uranyl acetate

stain. After a few seconds, this drop was also blotted dry and

the sample was examined under a Tecnai 12 (Bio-Twin

Transmission Electron Microscope; FEI, the Netherlands)

operating at 80 kV (Hayat & Miller, 1990).

Reactogenicity assay

The reactogenicity of the bacteria or the purified vesicles was

assayed by analysing interleukin (IL)-8 induction in HT-29

cells. Total RNA was extracted from the unstimulated cells

and their stimulated counterparts with TrizolTM reagent

(Invitrogen) following the manufacturer’s instructions.

cDNA was prepared from 2 mg of the extracted RNA using

Superscript II reverse transcriptase (Invitrogen), following

the standard protocol. IL-8 was amplified by SYBR-Greens

real-time quantitative PCR using ABI7300 (Applied Biosys-

tems). Relative quantitation was performed using the com-

parative threshold cycle number (CT) method. PCR was

performed with SYBR GreenTM Master MixTM (Applied

Biosystems), where SYBR green is the fluorescent reporter.

The internal control gene GAPDH was amplified simulta-

neously in separate reaction tubes. To eliminate primer

dimers, the fluorescent signal was collected at 82 1C, at

which temperature the primer dimmers melted, but the

PCR products were still in their double-stranded form,

thereby emitting fluorescence. The reaction conditions were

set as follows: initial heating at 95 1C for 5 min, followed by

40 cycles of reactions at 95 1C (1 min), followed by 62 1C

(2.5 min) and finally 82 1C (45 s). Final extension was

carried out at 62 1C for 8 min. The CT of triplicate reactions

was determined using ABI-SDS software and the mean CT of

triplicate reactions was determined. The levels of expression

of the genes of interest were normalized to GAPDH using

the formula 2�DDCT , where �DDCT is DCT (sample)�DCT

(calibrator) and DCT is the CT of the target gene subtracted

from the CT of the housekeeping gene (GAPDH). The

calibrator used in our experiments was the unstimulated

HT-29 cells.

Lipopolysaccharide preparation

Lipopolysaccharide prepared from different V. cholerae

strains was analysed by sodium dodecyl sulphate polyacry-

lamide gel electrophoresis (SDS-PAGE) (Hitchcock &

Brown, 1983). For lipopolysaccharide preparation, over-

night culture of V. cholerae in LB was harvested and washed

with PBS followed by treatment with 50 mL of lysis buffer

containing 2% SDS, 4% 2-mercaptoethanol, 10% glycerol,

1 M Tris (pH 6.8) and bromophenol blue. Lysates were

heated at 100 1C for 10 min. Proteins present in the lysates

were digested by adding 25mg of proteinase K solubilized in

10 mL of lysis buffer and heating the lysates at 60 1C for

60 min. Treated materials were subsequently electrophor-

esed onto SDS-PAGE and the polysaccharide materials were

fixed in the gel by treatment with ethanol–acetic acid

fixative. The gels were developed by silver staining to

visualize the lipopolysaccharide profile of various V. cholerae

strains.

Oral immunization of rabbits

New Zealand White rabbits of either sex (weighing 2–2.5 kg)

were immunized orally with OMVs (N16961) on days 0, 7,

14 and 21 according to the protocol developed by Spira et al.

(1981). The immunized group contained 30 rabbits and the

control group 18 rabbits (Tables 1 and 2). Rabbits were

fasted for 36 h, but water was given ad libitum. Thirty

minutes before the oral immunization, each rabbit was

anaesthetized by intramuscular injection of a mixture of

ketamine (35 mg kg�1 body weight; Sterfil Laboratories Pvt.

Ltd, India) and xylazine (5 mg kg�1 body weight, AstraZe-

neca Pharma India Ltd, India). Intravenous ranitidine

(50 mg kg�1 body weight; Ranbaxy, India) was administered

after 5 min, followed by two boluses of sodium bicarbonate

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd.

20 N. Roy et al.

(15 mL of a 5% solution; SRL, India) at 15-min intervals

introduced directly into the stomach through a feeding tube

(Romsons Sci. and Surg. Ind. Pvt. Ltd, India). The second

bolus was immediately followed by oral administration of

OMVs (50mg in 1 mL PBS) for the experimental rabbits and

the same volume of PBS for the control group. The rabbits

were returned to their cages and given limited amounts of

sterile food and water. Blood samples were collected for the

measurement of immunoglobulin titres before, during and

after immunization.

Serum collection

Blood was collected from the ear veins of rabbits on days 0,

7, 14, 21, 28, 35, 51, 65, 71 and 87. Collected blood was

allowed to clot at room temperature for 30 min and then

kept at 4 1C for 24 h. Serum was separated from the clotted

blood using a sterile Pasteur pipette and centrifuged

(10 min, 1000 g). The supernatant was collected and stored

at � 70 1C after addition of sodium azide to a final

concentration of 0.05% until further use.

Protection study

Postimmunization challenge of the rabbits with bacteria was

done following the protocol developed by Spira et al. (1981).

Homologous and heterologous (Tables 1 and 2) challenge

experiments were carried out on the 28th day after four

successive oral immunizations were completed with six

different strains of V. cholerae, as detailed above. Five

immunized and three unimmunized rabbits were challenged

with each strain. Briefly, all rabbits were fasted for 24 h

before the surgery and were provided water ad libitum.

Anaesthesia was performed with the mixture of ketamine

(50 mg kg�1) and xylazine (5 mg kg�1) administered intra-

muscularly. A midline incision 4–5 cm long was made

aseptically and the intestine was externalized at the level of

the ileocaecal valve. A caecal permanent tie was placed

1–2 cm from the ileocaecal junction using silk (Sadhana,

India). The exposed intestine was kept moist with sterile

saline. A removable slip-knot tie was placed on the ileum

10 cm proximal to the mesoappendix. A 1-mL (109) bacter-

ial inoculum was injected into the lumen of jejunum using a

sterile syringe and 25-G needle and the bowel was returned

to the abdominal cavity. The incision was closed and the

external free end of the tie was kept secured. The reversible

tie was released 4 h postinoculation and the rabbits were

kept on sterile water and food after recovery from anaes-

thesia. The rabbits were observed for 24 h for clinical signs of

diarrhoea, which was graded according to a system that

scores stool consisting of normal hard rabbit pellet as grade I

and a soft mushy stool as grade II. Catarrhal and loose

diarrhoea which took the form of the collection container

was scored grade III, while watery, liquid diarrhoeal stool

was grade IV.

Vibriocidal activity

Serum vibriocidal activity was measured according to the

method of Qadri et al. (1995) with minor modifications.

Vibrio cholerae strains grown to mid-logarithmic phase in

Table 2. Homologous and heterologous protection study of immunized rabbits in RITARD model

Experimental group Immunogen (OMV) Challenged strain

No. of animals

in each group Symptoms % death % protection

Immunized OMV N16961 5 Watery diarrhoea 0% (0/5) 0% (0/5) 100% (5/5)

Immunized OMV VC20 5 Watery diarrhoea 0% (0/5) 0% (0/5) 100% (5/5)

Immunized OMV 569B 5 Watery diarrhoea 0% (0/5) 0% (0/5) 100% (5/5)

Immunized OMV 0395 5 Watery diarrhoea 0% (0/5) 0% (0/5) 100% (5/5)

Immunized OMV SG24 5 Watery diarrhoea 40% (2/5) 0% (0/5) 60% (3/5)

Immunized OMV SG06 5 Watery diarrhoea 40% (2/5) 0% (0/5) 60% (3/5)

Table 1. Challenge study of control rabbits in RITARD model

Experimental group Immunogen (OMV) Challenged strain

No. of animals in

each group Symptoms % death % protection

Control PBS N16961 3 Watery diarrhoea 100% (3/3) 66.6% (2/3) 0% (0/3)

Control PBS VC20 3 Watery diarrhoea 100% (3/3) 0% (0/3) 0% (0/3)

Control PBS 569B 3 Watery diarrhoea 100% (3/3) 33.3% (1/3) 0% (0/3)

Control PBS 0395 3 Watery diarrhoea 100% (3/3) 0% (0/3) 0% (0/3)

Control PBS SG24 3 Watery diarrhoea 100% (3/3) 100% (3/3) 0% (0/3)

Control PBS SG06 3 Watery diarrhoea 100% (3/3) 66.6% (2/3) 0% (0/3)

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd.

21Vibrio cholerae OMVs vaccine candidate

TSB (Difco) were centrifuged, washed with PBS and resus-

pended to adjust the OD of the cell suspension to 0.20–0.22

at 600 nm wavelength. Freshly collected serum samples from

the immunized and the unimmunized rabbits were heat

treated for 1 h at 56 1C. Serum samples were serially diluted

in a 96-well plate and 50 mL of each dilution was used for

bactericidal activity. A reaction mixture was prepared using

PBS, bacterial cell and complement (Rockland) at a ratio of

6 : 3 : 1, and 50 mL of the reaction mixture was added to each

well containing the samples. After 1 h of incubation, 100mL

of brain heart infusion broth (BHI broth; Difco) was added

to each well of the 96-well plate and the reaction mixture

was incubated for further 4 h. For negative control reactions,

only PBS and complement was added. The positive control

contained PBS, bacterial cell, complement and El-tor, O1

Inaba, monoclonal antisera (Denka Seiken, Japan). The

reading was taken at 600 nm using an enzyme-linked

immunosorbent assay (ELISA) reader.

Immunoblot against lipopolysaccharide

Lipopolysaccharide of different V. cholerae strains was

resolved by SDS-PAGE and transferred to a nitrocellulose

membrane (Bio-Rad) at 0.25 A and 100 V for 1 h using a

transblot cell (Bio-Rad). The membrane was blocked with

5% skimmed milk (Bio-Rad) at room temperature for 2 h.

After washing with Tris-buffered saline (TBS) containing

0.1% Tween-20, the membrane was incubated with poly-

clonal rabbit antisera raised against OMVs of V. cholerae

N16961 strain at a dilution of 1 : 1000 for 90 min at room

temperature with gentle shaking. The nitrocellulose blot was

washed and incubated with a 1 : 5000 dilution of alkaline

phosphatase-conjugated antirabbit IgG (Sigma Chemical)

for 60 min at 37 1C. The blot was developed with a solution

of nitro blue tetrazolium and 5-bromo-4-chloroindolyl

phosphate.

ELISA

Analysis for the serum IgG antibody was performed using

ELISA, essentially following the method developed by Keren

(1979). Disposable polystyrene (Nunc, Denmark) microtitre

wells were coated with 10 mg of the whole cell lysate of V.

cholerae N16961 strain in 100mL of solution and incubated

for 18 h at 4 1C. Wells were washed three times with PBS-T

(PBS with 0.5% Tween-20). Nonspecific binding sites were

blocked by incubating the wells with 100 mL of 5% BSA

(Sigma Chemical) for 2 h at 37 1C. The wells were washed

three times with PBS-T and incubated with serially diluted

serum samples at 37 1C for 1 h. Following washing, 100mL

each of horseradish peroxidase-conjugated goat antirabbit

immunoglobulin G (Sigma Chemical) and the substrate o-

phenyl-D-amine (OPD) was added to each well and the plate

was incubated at 37 1C. The reaction was stopped after

10 min by adding 25 mL of 5 N sulphuric acid and the

reading was taken at 405-nm wavelength using an ELISA

reader.

Statistical analysis

Frequencies were compared by the chi-squared test, and the

continuous variable was compared using Student’s t-test.

P values � 0.05 and 0.01 were considered statistically

significant and highly significant, respectively.

Results

Vibrio cholerae produces OMVs

A 5-mL amount of OMVs was examined by transmission

electron microscopy, which showed multiple vesicle-like

structures representing OMVs. A few bacteria with vesicles

protruding from their surface were also seen and a repre-

sentative one is shown in Fig. 1.

Immunogenicity of OMVs

OMVs of several Gram-negative bacteria have been sug-

gested as candidates for vaccine development. To study the

immunogenic potential of V. cholerae OMVs, the serum IgG

antibody titre of rabbits against purified OMVs of the

N16961 strain was measured during the preimmunization,

immunization and postimmunization periods. The recipro-

cal titre of OMV-specific serum IgG increased linearly from

day 7 of immunization and reached a plateau around day 21.

This level was maintained during the remaining course of

immunization as well as during the postimmunization

period till the 51st day in most rabbits. In addition, a very

Fig. 1. Electron micrograph of free OMVs as well as those attached to

the bacteria (Vibrio cholerae N16961 strain) (� 20 magnification).

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd.

22 N. Roy et al.

high antibody titre was found until our final measurement

was taken 65 days after immunization was complete (Fig. 2).

Specificity of the antibody response

Bacterial lipopolysaccharide is a major immunogenic molecule

and the cause of the generation of vibriocidal antibodies in the

serum following infection/immunization. The lipopolysac-

charide profiles of the V. cholerae strains used in our studies

except SG24 were identical with respect to the rapid migrating

form. The SG24 strain lacked the long repetitive units of ‘O’

polysaccharide of lipopolysaccharide and was characterized by

the presence of the core oligosaccharide alone. Rabbit antibody

raised against the OMVs of N16961 strain could detect

lipopolysaccharide of several heterologous strains except that

of SG24 and SG06 in a Western blot analysis, suggesting

specificity for the ‘O’ polysaccharide (Fig. 3).

Protective efficacy of OMV sera

Vibriocidal antibody titre is considered to be the best

correlate and a surrogate marker of protection against V.

cholerae infection. Vibriocidal activity of serum is measured

by an in vitro complement fixation test that detects vibrio-

cidal antibody titres raised against the lipopolysaccharide of

V. cholerae. As the membrane of OMVs is derived from the

bacterial cell membrane and contains lipopolysaccharide, we

investigated whether the vesicles would induce vibriocidal

antibodies in rabbits. In our studies, rabbits were immu-

nized on days 0, 7, 14 and 21 and serum was collected on the

28th day to measure vibriocidal activities. While the im-

mune serum exhibited the highest vibriocidal activities

against the homologous strain, significant activities were

also found against most heterologous V. cholerae strains.

Among the latter group, the greatest vibriocidal potential

(�50%) was seen against VC20, 569B and O395 strains. In

contrast, only 22% and 15% killing was observed with SGO6

and SG24 strains, respectively (Fig. 4).

Protective efficacy of OMV immunization

To study the protective efficacy of OMV-specific serum

antibodies in vivo, rabbits were challenged with the homo-

logous and five of the heterologous V. cholerae strains

following oral immunization with OMVs (50mg) on days 0,

7, 14 and 21. On the 28th day, five immunized and three

unimmunized rabbits were challenged with each V. cholerae

strain as described under Materials and methods. Rabbits

were monitored for the next 24 h for the development of the

symptoms of ‘cholera’. All the unimmunized rabbits became

sick and developed moderate to profuse watery diarrhoea

within 16–18 h of challenge with bacteria, and their perineal

region remained constantly wet and soiled with faeces. In

contrast, two each (60%) from the immunized groups

challenged with SG24 and SGO6 strains, respectively, and

the rest that received a challenge with either of the four

V. cholerae strains developed no symptoms of diarrhoea and

were alive and healthy after 24 h (Tables 1 and 2). The above

results suggest 60–100% protective efficacies of the OMV

immunization schedule against the different V. cholerae

strains used in our study.

Reactogenicity of OMVs

Reactogenicity has remained a major bottleneck for many

vaccine strains of V. cholerae and has precluded their accep-

tance for mass vaccination despite considerable protective

efficacy. Reactogenicity is caused by the pro-inflammatory

Fig. 2. Vibrio cholerae OMVs are immunogenic. Five rabbits were

immunized orally on days 0, 7, 14 and 21 with OMVs (50 mg) purified

from V. cholerae N16961 strain. Serum IgG antibody titre was measured

on the days indicated and mean (� SD) values obtained from all the five

rabbits are presented here.

Fig. 3. OMV-induced serum antibody is lipopolysaccharide-specific.

(Left panel) SDS-PAGE of lipopolysaccharide extracted from different

Vibrio cholerae strains stained with Silver stain. (Right panel) Immunoblot

of lipopolysaccharide run in SDS-PAGE and probed with the rabbit

antiserum raised against OMVs of N16961. Lane 1: N16961, lane 2:

VC20, lane 3: 569B, lane 4: O395, lane 5: SG24 and lane 6: SGO6.

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd.

23Vibrio cholerae OMVs vaccine candidate

activities of the bacteria and predominantly results from the

induction of neutrophil chemotactic factor IL-8. To investi-

gate whether OMVs may also be reactogenic, human colon

epithelial cell line HT-29 was treated for 90 min with the

OMVs as well as the live, formalin-treated or heat-killed

bacteria. IL-8 mRNA expression in the cells was analysed by

qRT-PCR. The results showed that the live and the heat-

killed V. cholerae induced the highest levels of IL-8, whereas

the OMVs had the least effect (Fig. 5). These findings

indicate that a vaccine generated from OMVs may be less

reactogenic and better tolerated than the existing vaccines,

which are based on the live or the heat-killed cells.

Discussion

Gram-negative bacteria constitutively secrete OMVs into

the extracellular milieu. However, natural production of

large amounts of OMVs has only been described in a few

bacterial genera. Recent research in this area has revealed

that OMVs may act as intercellular communicasomes in

polyspecies communities by enhancing bacterial survival

and pathogenesis in hosts. Although research in this area

has revealed diverse functions of OMVs, the mechanisms of

vesicle formation and of protein sorting into OMVs, as well

as the pathophysiological role of OMVs, have not been

clearly defined (Lee et al., 2008).

Studies have shown that purified vesicles may induce

protective immune responses against subsequent infections

and may be potential candidates in vaccine development

(Henry et al., 2004). Camilli and colleagues successfully

purified large quantities of OMVs from two different strains

of V. cholerae (Schild et al., 2008). Here, we have purified

OMVs from a third strain, suggesting that many different

V. cholerae strains produce OMVs and that an OMV-based

vaccine may be useful against a wide range of V. cholerae

infections. Schild et al. (2009) used a mouse model to

demonstrate that purified OMVs administered through

oral, intranasal and intraperitoneal routes induce protective

immunity against colonization in the litters. Whereas pre-

natal transfer of anti-OMV immunoglobulins reduces

V. cholerae colonization, immunoglobulins transferred

through breast milk completely protect the neonates from

colonization (Schild et al., 2009). However, protection from

diarrhoea cannot be studied in a mouse model as mice do

not develop symptomatic cholera (Olivier et al., 2007). We

used the RITARD model, which closely resembles human

cholera, and showed that oral immunization with OMVs

completely prevents death and provides significant protec-

tion against the development of diarrhoea in rabbits. In

addition, we have demonstrated for the first time that

OMVs protect against the homologous as well as several

heterologous strains of V. cholerae. This is extremely im-

portant from a vaccination point of view, as different

V. cholerae serogroups and strains may be responsible for

diarrhoeal diseases not only in different geographical loca-

tions, but also at the same site at a particular point of time.

However, we have only used the oral route of immunization

in our studies. Although a previously published study

reported that equivalent protection was conferred by multi-

ple routes of immunization with OMVs (Schild et al., 2009),

it would be important to study the protective efficacy of

other routes besides oral immunization in rabbits. It is

noteworthy that OMVs induced long-term immunity in

both mouse and rabbit models.

Fig. 5. OMVs are the least reactogenic of the vaccines. The reactogeni-

city of different vaccine candidates was analysed by determining their

potency in inducing IL-8 in the cultured HT29 cells. Cells were stimulated

with formalin and heat-killed Vibrio cholera as well live bacteria and

purified vesicles for 90 min and IL-8 mRNA expression was analysed by

qRT-PCR (�Po 0.01, highly significant; 1Po 0.05, significant).

Fig. 4. Vibriocidal activity of OMV-induced antibody. Rabbits were

immunized with OMVs as described under Materials and methods.

Antiserum was collected on day 28 and the homologous and five

heterologous Vibrio cholerae strains were used for the serum vibriocidal

antibody assay as described above. Lane 1: N16961, lane 2: VC20, lane

3: 569B, lane 4: O395, lane 5: SG24 and lane 6: SGO6. The results were

expressed as fold increase compared with the nonimmune serum

(mean� SD of three independent experiments; �Po 0.01).

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd.

24 N. Roy et al.

Earlier studies suggested that the immunogenicity and

protective efficacy of OMVs may result from the proteins

(OMPs) (Cartwright et al., 1999; Borrow et al., 2005).

However, those authors did not investigate whether dena-

turation of the proteins resulted in loss of immunogenicity

and protective efficacy, as happens in the case of killed

whole-cell vaccines (Jertborn, 1992). OMPs may be respon-

sible for OMV-induced immunity in the case of Gram-

negative pathogens. However, the molecule of V. cholerae

that is mainly responsible for the induction of protective

immunity remains to be identified. Local protection in the

intestine is believed to be provided primarily by the secre-

tory IgA, and the serum vibriocidal antibody titre that is

induced by bacterial lipopolysaccharide is considered to be

the surrogate marker of protection. Our studies, for the first

time, strongly indicate that OMV-associated lipopolysac-

charide significantly induces vibriocidal antibody in the

serum. This is underscored by the fact that immunization

with OMVs of the N16961 strain gave rise to significant

vibriocidal antibody titres against heterologous V. cholerae

strains that possess a structurally similar lipopolysaccharide,

but not against the SG24 strain that has a different ‘O’

polysaccharide structure. Lack of response against SG24

strain could also result from the fact that V. cholerae O139

strains, as opposed to other serogroups, are encapsulated,

thus masking lipopolysaccharide from detection by antibo-

dies. Anti-OMV antibody indeed functions as a surrogate

marker of protection against subsequent V. cholerae infec-

tions as indicated by our studies, which showed that the

OMV-immunized rabbits were less protected against cho-

lera-like symptoms caused by the SG24 strains. It is not clear

from our studies whether OMV-specific IgG may directly

protect against gut infection, although previous studies have

suggested that serum IgG may be secreted in the intestine to

confer local protection (Ferrero et al., 1997).

Reactogenicity still remains a major obstacle in the

development of a vaccine that protects against V. cholerae

infection while at the same time causing minimal and

acceptable adverse reactions. Reactogenicity is fairly com-

mon with most vaccine strains of V. cholerae and is believed

to be caused by the proinflammatory reactions induced by

the bacteria. Although the bacterial molecule responsible for

this phenomenon remains to be identified, several virulence

factors may be functioning together, of which the motility-

associated protein flagellin may be the predominant inducer

of reactogenicity. IL-8 secreted by the intestinal mucosal

epithelial cells may be the initial signal for an acute

inflammatory response following bacterial encounter with

the host (Kagnoff & Eckmann, 1997). Recently, it was

suggested that there might be a correlation between the

reactogenicity and the ability of the V. cholerae strains to

induce IL-8 in vitro (Rodrıguez et al., 2001). We observed

significantly less IL-8 induction in the intestinal epithelial

cell line by the OMVs compared with the live bacteria and

the heat-killed whole cells, suggesting that an OMV-based

vaccine may be the least reactogenic vaccine.

Acknowledgements

This study was supported by a contingency grant from

NICED, ICMR. We thank Mr Krishna Biswas for animal

experimentation and Ms Tripti Biswas and Mr Suhasit

Ranjan Ghosh for technical assistance.

References

Alaniz RC, Deatherage BL, Lara JC & Cookson BT (2007)

Membrane vesicles are immunogenic facsimiles of Salmonella

typhimurium that potently activate dendritic cells, prime B and

T cell responses, and stimulate protective immunity in vivo. J

Immunol 179: 7692–7701.

Albert MJ, Alam K, Islam M, Montanaro J, Rahaman AS, Haider

K, Hossain MA, Kibriya AK & Tzipori S (1991) Hafnia alvei, a

probable cause of diarrhea in humans. Infect Immun 59:

1507–1513.

Balsalobre C, Silvan JM, Berglund S, Mizunoe Y, Uhlin BE & Wai

SN (2006) Release of the type I secreted alpha-haemolysin via

outer membrane vesicles from Escherichia coli. Mol Microbiol

59: 99–112.

Bauman SJ & Kuehn MJ (2006) Purification of outer membrane

vesicles from Pseudomonas aeruginosa and their activation of

an IL-8 response. Microbes Infect 8: 2400–2408.

Borrow R, Balmer P & Miller E (2005) Meningococcal surrogates

of protection-serum bactericidal antibody activity. Vaccine 23:

2222–2227.

Cartwright K, Morris R, Rumke H, Fox A, Borrow R, Begg N,

Richmond P & Poolman J (1999) Immunogenicity and

reactogenicity in UK infants of a novel meningococcal vesicle

vaccine containing multiple class 1 (PorA) outer membrane

proteins. Vaccine 17: 2612–2619.

Chatterjee SN & Chaudhuri K (2003) Lipopolysaccharides of

Vibrio cholerae. I. Physical and chemical characterization.

Biochim Biophys Acta 1639: 65–79.

Chatterjee SN & Das J (1967) Electron microscopic observations

on the excretion of cell-wall material by Vibrio cholerae. J Gen

Microbiol 49: 1–11.

Dalseg R, Wedege E, Holst J, Haugen IL, Høiby EA & Haneberg B

(1999) Outer membrane vesicles from group B meningococci

are strongly immunogenic when given intranasally to mice.

Vaccine 14: 2336–2345.

Davidsen T & Tonjum T (2006) Meningococcal genome

dynamics. Nat Rev Microbiol 4: 11–22.

de Moraes JC, Perkins BA, Camargo MC, Hidalgo NT, Barbosa

HA, Sacchi CT, Landgraf IM, Gattas VL, Vasconcelos HG &

Gral IM (1992) Protective efficacy of a serogroup B

meningococcal vaccine in Sao Paulo, Brazil. Lancet 340:

1074–1078.

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd.

25Vibrio cholerae OMVs vaccine candidate

Devoe IW & Gilchrist JE (1973) Release of endotoxin in the form

of cell wall blebs during in vitro growth of Neisseria

meningitidis. J Exp Med 138: 1156–1167.

Edwards JL & Apicella MA (2004) The molecular mechanisms

used by Neisseria gonorrhoeae to initiate infection differ

between men and women. Clin Microbiol Rev 17: 965–981.

Faruque SM, Albert MJ & Mekalanos JJ (1998) Epidemiology,

genetics and ecology of toxigenic Vibrio cholerae. Microbiol

Mol Biol R 62: 1301–1314.

Ferrero RL, Thiberge JM & Labigne A (1997) Local

immunoglobulin G antibodies in the stomach may contribute

to immunity against Helicobacter infection in mice.

Gastroenterology 113: 185–194.

Fiocca R, Necchi V, Sommi P, Ricci V, Telford J, Cover TL & Solcia

E (1999) Release of Helicobacter pylori vacuolating cytotoxin

by both a specific secretion pathway and budding of outer

membrane vesicles. Uptake of released toxin and vesicles by

gastric epithelium. J Pathol 188: 220–226.

Fredriksen JH, Rosenqvist E, Wedege E, Bryn K, Bjune G,

Frøholm LO, Lindbak AK, Møgster B, Namork E & Rye U

(1991) Production, characterization and control of

MenB-vaccine ‘Folkehelsa’: an outer membrane vesicle

vaccine against group B meningococcal disease. NIPH Ann 14:

67–79.

Gonzaleza S, Caballeroa E, Soriab Y, Cobasa K, Granadilloa M &

Pajona R (2006) Immunization with Neisseria meningitidis

outer membrane vesicles prevents bacteremia in neonatal

mice. Vaccine 24: 1633–1643.

Gorringe AR, Taylor S, Brookes C et al. (2009) Phase I safety and

immunogenicity study of a candidate meningococcal disease

vaccine based on Neisseria lactamica outer membrane vesicles.

Clin Vaccine Immunol 16: 1113–1120.

Hayat MA & Miller SE, eds (1990) Negative Staining, pp. 36–48.

McGraw-Hill, New York.

Henry T, Pommier S, Journet L, Bernadac A, Gorvel JP &

Lloubes R (2004) Improved methods for producing outer

membrane vesicles in Gram-negative bacteria. Res Microbiol

155: 437–446.

Hitchcock PJ & Brown TM (1983) Morphological heterogeneity

among polysaccharide lipopolysaccharide chemotypes in

silver-stained polyacrylamide gels. J Bacteriol 154: 269–277.

Hoekstra D, van der Laan JW, de Leij L & Witholt B (1976)

Release of outer membrane fragments from normally growing

Escherichia coli. Biochim Biophys Acta 455: 889–899.

Horstman AL & Kuehn MJ (2000) Enterotoxigenic Escherichia

coli secretes active heat-labile enterotoxin via outer membrane

vesicles. J Biol Chem 275: 12489–12496.

Jertborn M (1992) Cholera: transmission, treatment, prevention.

The 1991 epidemic is the first one in this century in Latin

America. Lakartidningen 89: 3942–3943, 3947.

Kadurugamuwa JL & Beveridge TJ (1999) Membrane vesicles

derived from Pseudomonas aeruginosa and Shigella flexneri can

be integrated into the surfaces of other Gram-negative

bacteria. Microbiology 145: 2051–2060.

Kagnoff MF & Eckmann L (1997) Epithelial cells as sensors for

microbial infection. J Clin Invest 100: 6–10.

Keenan J, Day T, Neal S, Cook B, Perez-Perez G, Allardyce R &

Bagshaw P (2000) A role for the bacterial outer membrane in

the pathogenesis of Helicobacter pylori infection. FEMS

Microbiol Lett 182: 259–264.

Keren DF (1979) Enzyme-linked immunosorbent assay for

Immunoglobulin G and Immunoglobulin A antibodies to

Shigella flexneri antigens. Infect Immun 24: 441–448.

Koley H, Ghosh AN, Paul M, Ghosh AR, Ganguly PK & Nair GB

(1995) Colonization ability & intestinal pathology of rabbits

orally fed with Vibrio cholerae O139 Bengal. Ind J Med Res 101:

57–61.

Kondo K, Takade A & Amako K (1993) Release of the outer

membrane vesicles from Vibrio cholerae and Vibrio

parahaemolyticus. Microbiol Immunol 37: 149–152.

Kuehn MJ & Kesty NC (2005) Bacterial outer outer membrane

vesicles and the host–pathogen interaction. Gene Dev 19:

2645–2655.

Lee EY, Choi DS, Kim KP & Gho YS (2008) Proteomics in Gram-

negative bacterial outer membrane vesicles. Mass Spectrom Rev

27: 535–555.

Mashburn-Warren LM & Whiteley M (2006) Special delivery:

vesicle trafficking in prokaryotes. Mol Microbiol 61: 839–846.

Mathan VI (1998) Diarrhoeal diseases. Brit Med Bull 54: 407–419.

Monroe SS, Stine SE, Gorelkin L, Herrmann JE, Blacklow NR &

Glass RI (1991) Temporal synthesis of proteins and RNAs

during human astrovirus infection of cultured cells. J Virol 65:

641–648.

Nevot M, Deroncele V, Messner P, Guinea J & Mercade E (2006)

Characterization of outer membrane vesicles released by the

psychrotolerant bacterium Pseudoalteromonas antarctica NF3.

Environ Microbiol 8: 1523–1533.

Olivier V, Salzman NH & Satchell KJ (2007) Prolonged

colonization of mice by Vibrio cholerae El Tor O1 depends on

accessory toxins. Infect Immun 75: 5043–5051.

Qadri F, Mohi G, Hossain J, Azim T, Khan AM, Salam MA, Sack

RB, Albert MJ & Svennerholm AM (1995) Comparison of the

vibriocidal antibody response in cholera due to Vibrio cholerae

O139 Bengal with the response in cholera due to Vibrio

cholerae O1. Clin Diagn Lab Immun 2: 685–688.

Richie EE, Punjabi NH, Sidharta YY et al. (2000) Efficacy trial of

single-dose live oral cholera vaccine CVD 103-HgR in North

Jakarta, Indonesia, a cholera-endemic area. Vaccine 18:

2399–2410.

Rodrıguez BL, Rojas A, Campos J, Ledon T, Valle E, Toledo W &

Fando R (2001) Differential interleukin-8 response of

intestinal epithelial cell line to reactogenic and nonreactogenic

candidate vaccine strains of Vibrio cholerae. Infect Immun 69:

613–616.

Schild S, Nelson EJ & Camilli A (2008) Immunization with Vibrio

cholerae outer membrane vesicles induces protective immunity

in mice. Infect Immun 76: 4554–4563.

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd.

26 N. Roy et al.

Schild S, Nelson EJ, Bishop AL & Camilli A (2009)

Characterization of Vibrio cholerae outer membrane vesicles as

a candidate vaccine for cholera. Infect Immun 77: 472–484.

Siadat SD, Kheirandish M, Norouzian D, Behzadiyannejad Q,

Najar Peerayeh S, Zangeneh M & Nejati M (2007) A flow

cytometric opsonophagocytic assay for measurement of

functional antibodies elicited after immunization with outer

membrane vesicle of Neisseria meningitidis serogroup B. Pak J

Biol Sci 10: 3578–3584.

Spira WM, Sack RB & Froehlich JL (1981) Simple adult rabbit

model for Vibrio cholerae and enterotoxigenic Escherichia coli

diarrhea. Infect Immun 32: 739–747.

Wai SN, Lindmark B, Soderblom T, Takade A, Westermark M,

Oscarsson J, Jass J, Richter-Dahlfors A, Mizunoe Y & Uhlin BE

(2003) Vesicle-mediated export and assembly of pore-forming

oligomers of the enterobacterial ClyA cytotoxin. Cell 115:

25–35.

FEMS Immunol Med Microbiol 60 (2010) 18–27

c� 2010 NICEDJournal compilation c� 2010 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd.

27Vibrio cholerae OMVs vaccine candidate