www.fems-microbiology.org

FEMS Immunology and Medical Microbiology 43 (2005) 425–430

Protective efficacy of a recombinant plague vaccine whenco-administered with another sub-unit or live attenuated vaccine

Kate Griffin a,*, Richard Bedford a, Kate Townson a, Robert Phillpotts a,Simon Funnell b, Margret Morton a, Diane Williamson a, Richard Titball a,c

a Defence Science and Technology Laboratories, Porton Down, Salisbury, Wiltshire SP4 0JQ, UKb Health Protection Agency, Porton Down, Salisbury SP4 0JG, UK

c Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

Received 10 June 2004; received in revised form 26 October 2004; accepted 28 October 2004

First published online 10 December 2004

Abstract

Vaccines against bioterrorism agents offer the prospect of providing high levels of protection against airborne pathogens. How-

ever, the diversity of the bioterrorism threat means that it may be necessary to use several vaccines simultaneously. In this study we

have investigated whether there are changes to the protective immune response to a recombinant sub-unit plague vaccine when it is

co-administered with other sub-unit or live attenuated vaccines. Our results indicate that the co-administration of these vaccines did

not influence the protection afforded by the plague vaccine. However, the co-administration of the plague sub-unit vaccine with a

live vaccine resulted in markedly increased levels of IgG2a subclass antibodies, and markedly reduced levels of IgG1 subclass anti-

bodies, to the plague sub-unit vaccine. This finding might have implications when considering the co-administration of other vaccine

combinations.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Co-administration; Plague; Vaccines

1. Introduction

Plague is a potentially fatal disease caused by the

gram-negative bacterium Yersinia pestis, which is usu-

ally transmitted to humans from infected animals viathe bite of an infected flea. Occasionally, plague is trans-

mitted from human to human via the airborne route.

Although the current worldwide incidence of plague is

low, regular small outbreaks do occur [1] and these have

the potential to develop into large scale outbreaks, as

demonstrated by the recent outbreak in India [2]. In

addition, Y. pestis has recently been highlighted as a

0928-8244/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2004.10.012

* Corresponding author. Tel.: +44 1980 613883; fax: +44 1980

614307.

E-mail address: kfgriffin@dstl.gov.uk (K. Griffin).

potential bioterrorism agent [3]. Although plague vac-

cines are of potential value in preventing disease the cur-

rently available killed whole cell vaccines (KWCV) may

cause a range of transient side-effects and the efficacy of

this formulation against the pneumonic form of the dis-ease has been questioned [4,5]. In order to address these

issues, sub-unit vaccines based on the protective proteins

Fraction 1 (F1) and V-antigen (V) are being developed

[4,5].

In the event of possible exposure of humans to bioter-

rorism or biological warfare agents, it is possible that

several vaccines would be used simultaneously. The is-

sues surrounding the use of vaccines in this way are verysimilar to those associated with the simultaneous admin-

istration of paediatric and travel vaccines. One obvious

advantage of vaccine co-administration is the reduction

. Published by Elsevier B.V. All rights reserved.

426 K. Griffin et al. / FEMS Immunology and Medical Microbiology 43 (2005) 425–430

in the number of visits required to receive all the neces-

sary immunisations, which often correlates with greater

user compliance. A number of paediatric vaccines are gi-

ven at the same time and research is underway to com-

bine more of the common infant vaccines into a single

injection [6–11]. However, immunisation with somecombinations of paediatric vaccines has resulted in sig-

nificant reductions in immunogenicity of individual vac-

cine components [12–16].

Multiple travel vaccines, such as those against hepa-

titis A, yellow fever and typhoid are also often co-ad-

ministered with no apparent impairment of the

immune response to the individual vaccines [17–20]. Re-

search into the tolerability of multiple travel vaccineschedules has suggested that the frequency of adverse

side effects, including transient local and systemic reac-

tions may increase with the number of vaccinations,

but that these side effects are generally mild [17,21,22].

In all cases, the advantages of multiple vaccination must

outweigh the possible concerns over variation in re-

sponses to the individual antigen components. An

acceptable multiple vaccination schedule must providecomparable efficacy and safety to its component vac-

cines. One issue affecting the co-administration of vac-

cines is whether the type of immune responses induced

interfere with one another, reducing protective efficacy.

Vaccines stimulate different types of immune response,

dependent on the type of infectious disease against

which they are directed. The preferential induction of

a T helper cell type 2 (Th2) response may be importantfor vaccines against extracellular pathogens and bacte-

rial exotoxins [23,24], whereas a T helper cell type 1

(Th1) response may be more important in combating

intracellular pathogens, such as viruses [25–27]. The im-

mune mechanisms induced by Th1 promoting vaccines

tend to upregulate type 1 responses and simultaneously

down-regulate type 2 responses and vice versa [28–32].

This mechanism of regulation allows bias of the immunesystem towards the appropriate mechanism of protec-

tion. Therefore, co-administration of vaccines designed

to promote different mechanisms may result in inappro-

priate regulation of immune responses causing a reduc-

tion in protective efficacy.

In this study we have immunised mice with a recomb-

inant plague vaccine formulation and investigated the

effect of co-administering vaccines that induce differenttypes of immune responses on anti-plague antibody re-

sponses and protective efficacy. One of the co-adminis-

tered vaccines we have used in this study was a live

vaccine against Venezuelan Equine Encephalitis, TC83,

which was selected as it stimulates Th1 responses [33]

and may therefore modulate the immune response to

the plague F1 + V vaccine. Two anthrax vaccines which

stimulate Th2 responses in mice were also used in thestudy; an experimental recombinant protective antigen

(PA) sub-unit vaccine [34] adjuvanted with Alhydrogel

and the current licensed Anthrax vaccine, a cell-free sup-

ernatant of the Sterne strain of Bacillus anthracis [35].

2. Materials and methods

2.1. Vaccines

The recombinant plague vaccine comprising the F1

and V antigen sub-units from Y. pestis has been de-

scribed previously [5,36]. The vaccine comprises 10 lgeach of the rF1 antigen and rV antigen sub-units ad-

sorbed to 25% v/v alhydrogel (Superfos Biosector, Den-

mark). The anthrax vaccine precipitate (AVP) is a cell-free supernatant produced from cultures of B. anthracis

Sterne strain [35] and was obtained from the Health Pro-

tection Agency, Porton Down, UK. The anthrax sub-

unit vaccine (rPA) has been described previously [34].

The vaccine comprises 10 lg of rPA adsorbed to 25%

v/v alhydrogel. Stocks of the Venezuelan Equine

Encephalitis (VEEV) vaccine TC-83 were prepared by

propagation from a vial of vaccine for human use (Na-tional Drug Company, Philadelphia, PA, Lot 4, run 2).

2.2. Immunisation regimens for co-administration of

vaccines

Groups of 10 adult 8–10-week-old female BALB/C

mice (Charles River Laboratories, UK) were immunised

as described in Table 1. Immunisation regimens were se-lected based on previous studies [5,32–37]. Immunisa-

tion with the rF1 + rV vaccine, the rPA vaccine and

the AVP vaccine were all carried out by the intramuscu-

lar route at the same site. Immunisation with TC-83 was

subcutaneous into the scruff.

2.3. Measurement of antibody responses

Serum samples were taken from groups of mice

immunised with the vaccines described in Table 1. Sam-

ples were assayed for the presence of antigen-specific

IgG1, IgG2a and IgG2b subclass antibody by ELISA.

Briefly, 96-well microtitre plates (Dyntech) were coated

with antigen diluted to 5 lg/ml in PBS. Non-specific

binding was blocked with 2% powdered skimmed milk

in PBS. Twofold dilutions of test samples were addedto plates in duplicate. Following a 2 h incubation at

37 �C, plates were washed with 0.02% Tween 20 in

PBS and horseradish peroxidase labelled, goat-anti

mouse IgG1, IgG2a or IgG2b antibody (Oxford Bio-

tech, UK) was added to wells. Following a further 2 h

incubation at 37 �C, plates were washed and the sub-

strate, 2,2 0-azino-di-3-ethylbenthiazoline sulphonate

(ABTS) was added. Twenty minutes after addition ofthe substrate, the A414 of the plates was measured. Anti-

body concentrations were calculated in ng/ml from

Table 1

Immunisation schedule

Primary immunisation Booster immunisation Challenge

rF1 + rV i.m. rF1 + rV i.m. day 21 Y. pestis (strain GB)

rF1 + rV i.m. and rPA i.m. rF1 + rV i.m. and rPA i.m. day 21 Y. pestis (strain GB)

rF1 + rV i.m. and AVP i.m. rF1 + rV i.m. and AVP i.m. day 21 Y. pestis (strain GB)

rF1 + rV i.m. and TC-83 s.c. rF1 + rV i.m. day 21 Y. pestis (strain GB)

Groups of 10 BALB/c mice were immunised as described above. Mice were challenged with Y. pestis strain GB 42 days after immunisation.

K. Griffin et al. / FEMS Immunology and Medical Microbiology 43 (2005) 425–430 427

standard curves generated with IgG antibody subclass

preparations of known concentration (Oxford Biotech,

UK) using Revelation� software (Dynex Technologies

Ltd., UK).

2.4. Y. pestis challenge

Y. pestis strain GB was originally isolated from afatal human case of plague and has a median lethal dose

(MLD) of <1 cfu in BALB/c mice by the subcutaneous

route [38]. Mice were challenged six weeks after the final

immunisation with 1 · 106 MLD of Y. pestis GB by the

subcutaneous route. The mice were observed for 14 days

for signs of illness and humane endpoints were used. All

animal experimentation strictly adhered to the Animals

(Scientific Procedures) Act 1986 and to the Guidance onthe Operation of the Animals (Scientific Procedures)

Act, as promulgated by the Home Office in the UK

and adopted by the ethics committee on animal experi-

mentation within this research establishment. All proce-

dures with live Y. pestis were performed under Advisory

Committee on Dangerous Pathogens (ADCP) category

three containment.

2.5. Statistical analysis

Values for the mean and the standard error of the

mean were calculated from the data. Unpaired Student�st test and Logrank Test were used to compare groups of

data. Probability values of <0.05 were taken as signifi-

cant. The results were analysed for statistical signifi-

cance with Prism 3 software (GraphPad Inc.).

3. Results and discussion

3.1. Antibody responses and protection afforded by a

recombinant plague vaccine in BALB/c mice

Previous work has shown that antibody which devel-ops after the immunisation of mice with rF1- and rV-an-

tigens plays a key role in protection against a subsequent

challenge with Y. pestis [39]. Other work has shown that

the titre of IgG1 antibody to these antigens correlates

with the degree of protection against Y. pestis challenge

[40]. When mice were immunised in this study with a

combination of 10 lg of rF1-antigen and 10 lg of rV-an-tigen they developed an antibody response which was

dominated by IgG1 class antibody against both proteins

(Fig. 1). When the immunised mice were subsequently

challenged with 106 MLD doses of Y. pestis strain GB

they did not show any signs of disease, and there were

no deaths by the end of the experiment. In contrast, con-

trol mice challenged with a similar dose of Y. pestis

developed disease and all animals had died by day 5 post

challenge.

3.2. Effect of co-administration of VEEV and anthrax

vaccines on antibody responses induced after

immunisation with a recombinant plague vaccine

Induction of the appropriate type of antigen-specificimmune response is critical for the success of vaccines.

The preferential induction of a type 2 response, charac-

terised by low levels of interferon-gamma (IFN-c) andhigh levels of interleukin-4 (IL-4), may be important

for vaccines against extracellular pathogens [23,24]. In

contrast, a type 1 response, characterised by high levels

of IFN-c and low levels of IL-4, may be more important

in combating intracellular pathogens such as viruses[25–27]. Antibodies are produced by either type of

response, but a Th1 induces B cells to produce the

complement-activating antibody IgG2a, whilst non-

complement-activating IgG1 is the predominant isotype

induced in a Th2 [41–43]. The vaccines used in this cur-

rent study were defined as inducing Th1 or Th2 re-

sponses by the predominant type of antibody and/or

the cytokine profile they induced. TC-83 is characterisedas a Th1 promoting vaccine [33] and rPA, AVP and

rF1 + rV are characterised as Th2 promoting vaccines

[36,44].

BALB/c mice were immunised with rF1 + rV alone or

in combination with rPA, AVP or TC-83. BALB/c mice

immunised with rF1 + rV alone developed high levels of

IgG1 antibody and low levels of IgG2a and IgG2b anti-

bodies to F1 and V antigens (Fig. 1). Co-administrationof rPA, had no effect on the anti-F1 and anti-V antibody

responses. In contrast, co-administration of AVP had no

effect on the level of IgG1 but significantly decreased the

anti-F1 and anti-V IgG2a and IgG2b responses

(p < 0.005). Although these differences were statistically

significant, the biological relevance of such low levels of

0

100

200

300

400

500

600

700

800

rF1+ rV rF1+ rV/rPA rF1+rV /AVP rF1+ rV/TC8 3

Treatment Group

IgG

1 (u

g/m

l)

An ti-V

An ti-F 1

*

***

0

20

40

60

80

100

120

140

160

180

200

rF 1+ rV rF 1+ rV /r PA rF 1+ rV /AVP rF 1+ rV /T C8 3

Treatment Group

IgG

2a (

ug

/ml)

Anti -V

Anti -F1

*** *

** *

** *

0

2

4

6

8

10

12

rF 1+ rV rF 1+ rV /rPA rF 1+ rV /AVP rF 1+ rV /T C8 3

Treatment Group

IgG

2b (

ug

/ml)

Anti-V

Anti-F1

** ** **

******

(a)

(b)

(c)

Fig. 1. (a–c): Anti-F1 and anti-V IgG subclasses produced in response to co-administering the plague subunit vaccine with the anthrax subunit

vaccine, the AVP vaccine and the VEEV. The effect of vaccine co-administration was determined by comparing the responses to administration of the

plague subunit vaccine alone. *p < 0.01, ***p < 0.001.

428 K. Griffin et al. / FEMS Immunology and Medical Microbiology 43 (2005) 425–430

IgG2a and IgG2b was not determined. The greatest ef-

fect on antibody responses was seen following the co-ad-

ministration of the TC-83 vaccine with rF1 and rV.

Anti-F1 and anti-V antigen IgG2a and 2b responses sig-nificantly increased (p < 0.0001 for anti-V and anti-F1

titres) whilst IgG1 responses significantly decreased

(p < 0.0001 for anti-V antigen titres; p = 0.0032 for

anti-F1 antigen titres). In previous studies similar results

were obtained in both BALB/c and C57/BL6 strains of

mice (data not shown) indicating that this was not a

haplotype specific effect.

3.3. Effect of co-administration of VEEV and anthrax

vaccines on protective responses induced after

immunisation with a recombinant plague vaccine

Previously it has been shown that the combined titre

of IgG1 subclass antibodies to F1 and V-antigens corre-

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14012

345

6789

10

rF1+ VrF1+ V/rPArF1+ V/AVPrF1+ V/TC -83

non-immunised

Timepost challenge (days)

Nu

mb

er o

f su

rviv

ors

Fig. 2. Protection against Y. pestisGB challenge. Groups of 10 BALB/

c mice were immunised and the effect of co-administration on rF1 + V

efficacy was assessed. The graph shows the number of animals

surviving subcutaneous challenge with 1 · 106 MLD Y. pestis.

K. Griffin et al. / FEMS Immunology and Medical Microbiology 43 (2005) 425–430 429

lates with protection against plague in the murine model

of disease [40]. Since the co-administration of TC83 vac-

cine with the recombinant plague vaccine had a pro-

found influence on the type of antibody response, weinvestigated whether this altered response would protect

against challenge with Y. pestis. Mice were immunised

with the rF1 and rV vaccine and challenged six weeks la-

ter with 106 MLD doses of Y. pestis by the subcutaneous

route. Control (unimmunised) mice had all died by day

5, whereas all of the immunised mice showed no signs of

disease and survived, irrespective of whether they had

also received the TC-83 vaccine (Fig. 2). The results ob-tained in this study show that the decrease seen in IgG1

following co-administration of TC-83 was not sufficient

to cause a reduction in protection. It is possible that

although the IgG1 level had declined in the co-adminis-

tered group this was not below a threshold at which pro-

tection is lost, even to such a high microbiological

challenge. However, it is also possible that IgG1 is not

the only effector of protection which develops afterimmunisation with the rF1 + rV vaccine. Some previous

work on plague vaccine formulations based on V anti-

gen have shown clearly that an antigen-specific IgG1 re-

sponse is not always indicative of protection [45]. This

study has concentrated primarily on the effect of co-ad-

ministration on antibody response since antibody in-

duced by the vaccine formulation used has been

clearly identified as a protective immune effector mecha-nism in this model.

Our findings suggest that immunisation with other

vaccine combinations may well result in the modulation

of immune responses. Of particular significance is the

finding that although the administration of a Th2 biased

vaccine with a live attenuated vaccine, even at different

sites, results in an antibody profile which is indicative

of a Th1 biased response, protective efficacy was not im-paired. Further work is required to investigate the pos-

sible impact of co-administration on both live and

sub-unit Th1 biased vaccines.

Acknowledgements

The authors thank Tony Stagg, Debbie Rogers, Deb-

bie Bell and Dave Rawkins for technical assistance.

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