Published Ahead of Print 27 July 2011. 

10.1128/CVI.05133-11. 

2011, 18（9）:1497. DOI: Clin. Vaccine Immunol. 

Puyan Chen

Deyuan Li, Maoyun Xue, Chen Wang, Junbao Wang and

Avian Influenza Virus in Chickens

Immune Responses to Inactivated H9N2

Enhances both Humoral and Cell-Mediated 

Bursopentine as a Novel Immunoadjuvant

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 on April 26, 2012 by Huazhong Agricultural University http://cvi.asm.org/ Downloaded from CLINICAL AND VACCINE IMMUNOLOGY, Sept. 2011, p. 1497–1502 Vol. 18, No. 9

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Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Bursopentine as a Novel Immunoadjuvant Enhances both Humoral

and Cell-Mediated Immune Responses to Inactivated H9N2

Avian In?uenza Virus in Chickens

Deyuan Li,

1

†* Maoyun Xue,

2

† Chen Wang,

3

Junbao Wang,

4

and Puyan Chen1

Division of Key Lab of Animal Disease Diagnosis and Immunology, China’s Department of Agriculture, Nanjing Agricultural

University, Nanjing 210095, People’s Republic of China1

; Food Safety Technological Engineering Research Centre,

Jiangsu Institute of Economic and Trade Technology, Nanjing 210007, People’s Republic of China2

;

College of Animal Science and Technology, Henan University of Science and Technology,

Luoyang 471003, People’s Republic of China3

; and Dandong National School,

Eastern Liaoning University, Dandong 118005, People’s Republic of China4

Received 3 May 2011/Returned for modi?cation 6 May 2011/Accepted 18 July 2011

There is an urgent need for identi?cation of a new adjuvant capable of selectively promoting an ef?cient

immune response for use with vaccines and especially subunit vaccines. Our pervious study showed that

Bursopentine （BP5） is a novel immunomodulatory peptide and has the ability to signi?cantly stimulate an

antigen-speci?c immune response in mice. In this study, the potential adjuvant activities of BP5 were examined

in chickens by coinjection of BP5 and an inactivated avian in?uenza virus （AIV） （A/Duck/Jiangsu/NJ08/05

[AIV H9N2 subtype]）. The results suggested that BP5 markedly elevated serum hemagglutination inhibition

（HI） titers and antigen-speci?c antihemagglutinin （anti-HA） antibody （IgG） levels, induced both Th1 （inter-

leukin 2 [IL-2] and gamma interferon [IFN-]）- and Th2 （IL-4）-type cytokines, promoted the proliferation of

peripheral blood lymphocytes, and increased populations of CD3 T cells and their subsets CD4 （CD3

CD4） T cells and CD8 （CD3 CD8） T cells. Furthermore, a virus challenge experiment revealed that BP5

contributes to protection against homologous avian in?uenza virus challenge by reducing viral replication in

chicken lungs. This study indicates that the combination of inactivated AIVs and BP5 gives a strong immune

response at both the humoral and cellular levels and implies that BP5 is a novel immunoadjuvant suitable for

vaccine design.

The immune-promoting activity of any given vaccination

strategy is set not only by the presence of the relevant antigenic

components in the vaccine formulation but also by the com-

plement of suitable adjuvants （9, 20）. When incorporated into

a vaccine formulation, a suitable adjuvant acts to accelerate,

extend, or enhance the magnitude of a speci?c immune re-

sponse to the vaccine antigen （6）. Strategies for improving

current vaccines have emphasized making currently available

vaccines more ef?cacious by developing a better adjuvant, es-

pecially for inactivated viral and subunit vaccines.

Bursopentine （BP5; with an amino acid sequence of Cys-

Lys-Asp-Val-Tyr） is a novel immunomodulatory peptide iso-

lated from chicken bursa of Fabricius （19）. As it has the ability

to signi?cantly stimulate antigen-speci?c immune responses at

both the humoral and cellular levels in mice immunized with

inactivated avian in?uenza viruses （AIVs） （19）, its potential

adjuvant activities were assessed in chickens in this study by

using a model antigen of an inactivated AIV, A/Duck/Jiangsu/

NJ08/05 （AIV H9N2 subtype）.

In many countries, H9N2 AIVs are an enormous economic

burden on the commercial poultry industry when they cause

signs of mild respiratory disease and a reduction in egg pro-

duction. In April 1999, two World Health Organization refer-

ence laboratories independently con?rmed the isolation of

avian in?uenza A （H9N2） viruses for the ?rst time in humans

（39）. An increased risk of direct transmission of these viruses

to humans is possible （21, 25, 29）. Inactivated vaccines have

been used to control AIV infection, but the best protection

against AIV infection remains effective vaccination. Previ-

ously, it has been shown that inactivated vaccines elicit strong

humoral responses, and it is commonly accepted that no ade-

quate mucosal or cellular immunity is achieved （37）. However,

cellular immunity is essential for virus clearance at the end

stage of many viral infections （4）. Adjuvants are able to im-

prove the quantity and quality of innate immune responses by

enhancing their speed and duration and by inducing adequate

adaptive immunity （31）. In the current study, BP5 was used as

an adjuvant for our AIV vaccination strategy to provide an

effective way to prevent and control H9N2 AIV infection.

The effect of BP5 on humoral and cell-mediated immune

responses induced by inactivated AIV vaccination was evalu-

ated in 1-week-old speci?c-pathogen-free White Leghorn

chickens. Humoral immunity was measured by detection of

antigen-speci?c antibody titer and antihemagglutinin （anti-

HA） IgG responses using the hemagglutination inhibition （HI）

test and enzyme-linked immunosorbent assay （ELISA）, re-

spectively. Cell-mediated immunity was evaluated by detection

* Corresponding author. Mailing address: Division of Key Lab of

Animal Disease Diagnosis and Immunology, China’s Department of

Agriculture, Nanjing Agricultural University, Nanjing Agricultural

University, 1 Weigang, Nanjing, JingSu 210095, China. Phone: 86-25-

84396028. 86-25-84396335. : deyuanlinjau.

† Deyuan Li and Maoyun Xue contributed equally to the paper.

Published ahead of print on 27 July 2011.

1497

 on April 26, 2012 by Huazhong Agricultural University http://cvi.asm.org/ Downloaded from of serum Th1 （interleukin 2 [IL-2] and gamma interferon

[IFN-]）- and Th2 （IL-4）-type cytokines （23） by ELISA, by

measurement of chicken peripheral blood lymphocyte prolif-

eration using the 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-

tetrazolium bromide （MTT） assay, and by measurement of

chicken peripheral blood CD3 T cells and their subsets CD4

（CD3 CD4） T cells and CD8 （CD3 CD8） T cells by an

immunophenotyping assay. Furthermore, virus challenge ex-

periments were assayed to evaluate the protection of activated

AIV vaccine administered with BP5 against homologous avian

in?uenza virus replication in chicken lungs.

MATERIALS AND METHODS

Preparation of BP5. Synthetic BP5 was purchased from Shanghai Biotech

Bioscience and Technology Co., Ltd. （People’s Republic of China）. The se-

quence of the synthetic peptide was con?rmed by electrospray ionization tandem

mass spectrometry （ESI-MS/MS）, and the purity of the synthetic peptide was

98% by reversed-phase high-performance liquid chromatography （RP-HPLC）.

Viruses and chicken. AIV A/Duck/Jiangsu/NJ08/05 （AIV H9N2 subtype） was

provided by the Institute of Animal Husbandry and Veterinary Medicine, Ji-

angsu Academy of Agricultural Sciences （Nanjing, China）. Avian in?uenza

H9N2 virus strain JS-1 （A/Chicken/Jiangsu/JS-1/2002） was isolated and kept in

our own laboratory. AIVs were cultured in the allantoic sacs of chicken embryos.

The AIV hemagglutination titer of the inoculated allantoic ?uid was 1:210

,

corresponding to 107

50% tissue culture infective doses （TCID50）/0.1 ml. The

A/Duck/Jiangsu/NJ08/05 AIV was inactivated with 0.025% formaldehyde for

72 h at 4°C. Its ef?cacy was tested by three blind virus passages in speci?c-

pathogen-free （SPF） eggs （19, 38）, and the inactivated AIV was used as a vaccine

antigen for the following experiments.

One-week-old SPF White Leghorn chickens from Qian Yuan hao Co., Ltd.

（Nanjing, China）, were obtained as fertilized eggs, hatched, and maintained

in an isolation facility at the Poultry Research Institute （Nanjing, China）. All

groups of chickens were housed, handled, and immunized in accordance with

the guidelines and with the approval of the local institutional animal exper-

iment committee.

Vaccination of chickens. SPF White Leghorn chickens were randomly divided

into six experimental groups of 18 chickens each and intramuscularly immunized

two times on days 0 and 14 with （i） a mixture of 400 l AIV （A/Duck/Jiangsu/

NJ08/05, 107

TCID50/0.1 ml） and 100 l phosphate-buffered saline （PBS）, （ii） a

mixture of 400 l AIV （A/Duck/Jiangsu/NJ08/05, 107

TCID50/0.1 ml） and 25, 5,

or 1 mg BP5 in 100 l PBS/kg body weight, （iii） 400 l commercially inactivated

AIV/H9N2 vaccine （an oil-formulated vaccine obtained from Qian Yuan hao

Co., Ltd., Nanjing, China [107

TCID50/0.1 ml]） plus 100 l PBS as a positive

control, or （iv） 500 l PBS as a negative control （Table 1）.

HI assay. On days 14 and 28 postimmunization, serum hemagglutination

inhibition （HI） antibody titers of each group of chickens were evaluated with an

HI test based on Hirst’s principle （10）. The serum was diluted 10-fold with saline

before an additional 2-fold dilution with PBS was made. Standard avian in?uenza

antigen （Harbin Veterinary Research Institute, China） with 4 hemagglutination

units was then added to each diluted serum sample and mixed for approximay

15 min. An equal volume of 0.5% chicken red blood cells was added to the

virus-serum mixture and incubated for 30 to 60 min before the results were read.

The HI titers were de?ned as the highest serum dilution capable of preventing

hemagglutination.

Estimation of antigen-speci?c antibodies （IgG）. Sera from chickens were

collected on days 14 and 28 postimmunization. Speci?c antihemagglutinin （anti-

HA） IgG of chicken sera was analyzed by ELISA. Brie?y, ELISA plates were

coated with a puri?ed prokaryote-expressed recombinant JS-1 （A/Chicken/

Jiangsu/JS-1/2002, H9N2 AIV） HA protein （preserved in our laboratory, 10

g/ml） （40）. Serially diluted chicken sera were then incubated for2hat room

temperature, followed by a 1-h incubation with horseradish peroxidase （HRP）-

conjugated goat anti-chicken IgG （GenScript Co., Ltd., China）. Titers at half-

maximal optical densities （OD） were determined by linear interpolation between

the measured points neighboring the half-maximal OD. Linear interpolation was

calculated using the logarithm of the titer values. Each serum titration was

repeated in triplicate.

Cytokine assays. One day 28 postimmunization, the serum levels of Th1-type

cytokines （IL-2 and IFN-） in chickens were determined with commercial

ELISA kits （Cusabio Biotech）, whereas Th2-type cytokine （IL-4） was measured

by another commercial ELISA kit （R&D Systems, United Kingdom）. The pro-

cedure followed the manufacturer’s instructions.

Lymphocyte proliferation assay and immunophenotyping assay. To detect

changes in cellular immunity, a peripheral blood lymphocyte proliferation assay

and an immunophenotyping assay were performed. Fourteen days after the

second immunization （day 28）, the blood samples were collected for lymphocyte

separation. Peripheral blood lymphocytes were separated as described previ-

ously, with some modi?cation （11, 28）. The cell suspension from the blood was

layered on Ficoll-Paque lymphocyte separation medium by density gradient

centrifugation. Peripheral blood lymphocytes were obtained from the interface

and washed twice with Hanks’ balanced salt solution. After centrifugation, the

?nal pellet was resuspended in RPMI 1640 medium containing 5% heat-inacti-

vated fetal calf serum at a concentration of 2 106

cells per ml.

The peripheral blood lymphocyte proliferation assay was performed using a

modi?ed MTT method as described previously （13, 22）. Brie?y, the peripheral

blood lymphocytes （2 106

cells/ml） were dispersed and incubated in 96-well

?at-bottomed microtiter plates （80 l/well）. Another 20 l of concanavalin A

（ConA; 10 g/ml, positive control）, the recombinant JS-1 （A/Chicken/Jiangsu/

JS-1/2002, H9N2 AIV） HA protein （10 g/ml, speci?c antigen stimulation）, or

RPMI 1640 medium without antigen （negative control） was added to each well,

and each sample was seeded in four wells. After 44 h of incubation at 39.5°C in

a5%CO2 incubator, 20 l of MTT （dissolved in PBS, 5 mg/ml） （Sigma） was

added to each well and the incubation was continued for another 4 h. Then 100

l of dimethyl sulfoxide （DMSO） was added, and incubation was continued for

an additional 24 h before measurement of OD at 750 nm （OD570） using an

ELISA reader （Bio-Tek Instruments, VT）. Cell viability is expressed as the

percentage of the OD570 of cells treated with complex over the OD570 of the

control samples.

Flow cytometric analysis of peripheral blood lymphocytes was carried out as

previously described （30）. Peripheral blood lymphocytes （2 106

cells/ml） were

made complex with the monoclonal antibody phycoerythrin （PE）-labeled anti-

chicken CD3 and then with PE-labeled anti-chicken CD4 and ?uorescein

isothiocyanate （FITC）-conjugated anti-chicken CD8 （Southern Biotechnology）

for1hat 4°C. PE- and FITC-conjugated isotype controls were also included.

Cells were analyzed by ?uorescence-activated cell sorting （BD Biosciences）.

Virus challenge experiment. Two weeks after the second vaccination, all chick-

ens were intranasally challenged with 2 107

TCID50 of avian in?uenza H9N2

virus strain JS-1 （A/Chicken/Jiangsu/JS-1/2002） in 0.1 ml. Lungs were collected

from six chickens from each group at 1, 3, and 5 days after virus challenge （Table

1）. All lung samples were stored at 80°C. Viral copy numbers in lungs were

determined by using real-time PCR. An RNeasy RNA extraction kit （Invitrogen,

Norway） was used to prepare total RNA from the lung samples. The RNA was

reverse transcribed to cDNA by using the reverse transcription system from

Promega （Germany）. A 2-l portion of cDNA was used to amplify the HA gene

by real-time PCR using one pair of PCR primers: HA- forward, 5-CTACTGT

TGGGAGGAAGAGAATGGT-3, and HA-reverse, 5-TGGGCGTCTTGAAT

AGGGTAA-3. PCR primers were designed based on the HA gene sequence of

avian in?uenza H9N2 virus strain JS-1 （A/Chicken/Jiangsu/JS-1/2002） in

GenBank （accession no. AY364228）. The ampli?cation was performed by using

SYBR green （ABI, Warrington, United Kingdom） according to the method

described previously （24）, with some modi?cations. The standard curve for

real-time PCR quanti?cation was constructed using the HA gene in the vector

pET32a-HA （H9N2）, a gift from Qisheng Zheng （Institute of Veterinary Sci-

ence, Jiangsu Academy of Agricultural Sciences）. The pretreatment of the re-

TABLE 1. Animal groups and the experimental designa

Group Vaccination on days 0 and 14b

1 ..........................500 l PBS

2 ..........................400 l AIVs （107

TCID50/0.1 ml） 100 l PBS

3 ..........................400 l AIVs （107

TCID50/0.1 ml） 100 l BP5

（25 mg/kg/0.1 ml PBS）

4 ..........................400 l AIVs （107

TCID50/0.1 ml） 100 l BP5

（5 mg/kg/0.1 ml PBS）

5 ..........................400 l AIVs （107

TCID50/0.1 ml） 100 l BP5

（1 mg/kg/0.1 ml PBS）

6 ..........................400 l H9N2 AIV vaccine （107

TCID50/0.1 ml）

100 l PBS

a

All chickens were challenged on day 28.

b

AIVs, inactivated H9N2 avian in?uenza virus; H9N2 AIV vaccine, commer-

cial H9N2 avian in?uenza virus vaccine prepared with oil/water as an adjuvant.

1498 LI ET AL. CLIN.VACCINE IMMUNOL.

 on April 26, 2012 by Huazhong Agricultural University http://cvi.asm.org/ Downloaded from action mixture was carried out at 94°C for 10 min, and then the mixture was

subjected to 40 cycles of ampli?cation at 95°C for 15 s and at 60°C for 30 s.

Statistical analysis. Antibody titers, cytokine levels, percentages re?ecting

lymphocyte proliferation, percentages of CD3, CD3 CD4, and CD3 CD8

cells in the peripheral blood, and numbers of viral copies in chicken lungs were

recorded as means standard deviations （SD）. Bonferroni correction multiple-

comparison tests were used to evaluate any differences between groups. Differ-

ences between means were considered signi?cant at a P of 0.05 or a P of 0.01.

RESULTS

BP5 stimulates signi?cant antigen-speci?c immune re-

sponses. To test antigen-speci?c immune responses to immu-

nization, chickens were immunized two times with a mixture of

BP5 and inactivated avian in?uenza viruses （AIVs） or a com-

mercial AIV （H9N2） vaccine （positive controls） or with PBS

（negative control）. Chickens coimmunized with inactivated

AIVs and BP5 produced signi?cantly higher hemagglutination

inhibition （HI） antibody titers （Fig. 1A） （after priming with 25,

5, and 1 mg/kg [P 0.05 {*}] and boosting with 25 and 5 mg/kg

[P 0.05 {#}] and 1 mg/kg [P 0.01 {##}]） and anti-HA

antibody （IgG） titers （Fig. 1B） （after priming with 25 and 5

mg/kg [P 0.05 {*}] and 1 mg/kg [P 0.01 {**}] and boosting

with 25 and 5 mg/kg [P 0.05 {#}] and 1 mg/kg [P 0.01

{##}]） than those immunized with inactivated AIVs alone.

Compared to chickens immunized with the commercial H9N2

AIV vaccine （with a combination of oil and water [oil/water] as

an adjuvant）, chickens coadministered inactivated AIVs and

BP5 also produced signi?cantly higher HI （Fig. 1A） and IgG

（Fig. 1B） antibody titers （after priming with 1 mg/kg [P 0.05

{†}] and boosting with 5 mg/kg and 1 mg/kg [P 0.05 {§}]）.

BP5 increases the production of both Th1- and Th2-type

cytokines. We then tested the levels of Th1 （IL-2 and IFN-）

and Th2 （IL-4） cytokines upon coimmunization with inacti-

vated AIV and BP5 in chickens. Compared with restimulation

with inactivated AIVs alone, coimmunization with inactivated

AIVs and BP5 remarkably increased the levels of both Th1-

type （IL-2 and IFN- [P 0.05]） and Th2-type （IL-4 [P

0.01]） cytokines in chickens, whereas only Th1-type cytokines

increased with commercially inactivated H9N2 AIV vaccine

restimulation （Fig. 2）.

BP5 signi?cantly enhances peripheral blood lymphocyte

proliferation. To investigate the effects of BP5 on peripheral

lymphocyte proliferation, we collected peripheral lymphocytes

from chickens treated with different dosages of BP5 coadmin-

istered with inactivated AIV and treated them with recombi-

nant JS-1 （A/Chicken/Jiangsu/JS-1/2002, H9N2 AIV） HA pro-

tein in vitro. When chickens were immunized with inactivated

AIVs and BP5, a signi?cant proliferative response was ob-

served （Fig. 3; *, P 0.05, compared with chickens immunized

with the inactivated AIVs alone; #, P 0.01, compared with

chickens immunized with PBS; †, P 0.05, compared with

chickens immunized with the commercially prepared H9N2

AIV vaccine with oil/water as an adjuvant）. The data showed

that chickens immunized with a combination of BP5 and inac-

tivated AIVs also induce the highest AIV-speci?c cellular pro-

liferation, in addition to the humoral responses described

above.

FIG. 1. Effects of adding BP5 to the inactivated AIVs on the levels

of antigen-speci?c HI titers （A） and anti-HA IgG antibodies （B）.

Chicken sera were collected on days 21 and 28 postimmunization, and

the serum HI titers and IgG titers were analyzed by HI assay and by

ELISA, respectively. The data presented are means SD of results

from three replicates. , P 0.05, and , P 0.01 （prime）; #, P

0.05, and ##, P 0.01 （boost）, compared to chickens immunized with

inactivated AIVs alone. †, P 0.05 （prime）; §, P 0.05 （boost）,

compared to chickens immunized with the commercial H9N2 AIV

vaccine.

FIG. 2. Effect of adding different doses of BP5 to inactivated AIVs

on Th1/Th2 cytokine production in chicken sera. Chickens were im-

munized two times, and chicken sera were collected on day 28 postim-

munization. Cytokine release was measured by using a sandwich

ELISA method and commercial ELISA kits. The data presented are

means SD of results from four replicates. , P 0.05; , P 0.01,

compared to chickens immunized with AIVs alone. †, P 0.01, com-

pared to chickens immunized with the commercial H9N2 AIV vaccine.

VOL. 18, 2011 BURSOPENTINE （BP5） AS A NOVEL IMMUNOADJUVANT 1499

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ages of overall CD3 T cells and their subsets （CD4 T cells

[CD3 CD4] and CD8 T cells [CD3 CD8]） in the pe-

ripheral blood lymphocyte populations were signi?cantly in-

creased in the chickens immunized with a mixture of inacti-

vated AIV and BP5 （5 mg/kg, P 0.05; 1 mg/kg, P 0.01）

compared with those in chickens immunized with inactivated

AIV alone （Table 2）. However, CD8 T cells were only mod-

eray affected by administration of the commercial H9N2

AIV vaccine. This indicated that BP5 has an adjuvant activity

in that it promotes the AIV vaccine by stimulating not only

CD4 T cell proliferation but also CD8 T cell proliferation.

BP5 signi?cantly promotes immune protection against

H9N2 AIV challenge. To verify that a killed vaccine in combi-

nation with BP5 can provide better protection against H9N2

AIV infection, we applied a real-time PCR assay using SYBR

green 1 for detection of AIV copies in the lungs of chickens on

days 1, 3, and 5 after H9N2 AIV challenge. In the assay, the

dissolution curve showed that the HA primer had a good

speci?city, and the standard curve results showed that the

ampli?cation ef?ciency of the HA primer, which could be used

for detection of virus in lung samples, was 99.89% （data not

shown）. As shown in Table 3, numbers of lung viral copies were

signi?cantly reduced in the chickens coimmunized with inacti-

vated AIVs and BP5 compared to those in the chickens im-

munized with inactivated AIV alone on days 1, 3, and 5 after

H9N2 AIV challenge （25 mg/kg and 5 mg/kg, P 0.05; 1

mg/kg, P 0.01）. Compared to the number of lung viral copies

in the chickens immunized with the commercial H9N2 AIV

vaccine （with oil/water as an adjuvant）, lung viral copies were

also reduced signi?cantly in the chickens coadministered inac-

tivated AIVs and BP5 （1 mg/kg, P 0.05） （Table 3）.

DISCUSSION

Many adjuvant approaches have been evaluated for use in

vaccines. However, since most of the adjuvants used in conju-

gation with antigen have unacceptable levels of side effects,

such as toxicity and adverse site reactions, only a few of them

are used clinically （26, 35）. Aluminum-based mineral salts （alu-

minum adjuvant; alum） have commonly been used in many

veterinary and human vaccines because of their safety （1）, but

they induce antibody production weakly and are poor at elic-

iting cell-mediated immune responses （3）, which are signi?cant

drawbacks for their use in vaccines against intracellular para-

sites and some viruses. The oil-based adjuvants, which are

common in veterinary vaccines, in contrast, are limited by their

induction of side effects and adverse site reactions （5, 18, 34）.

Thus, research to ?nd new and optimal adjuvant candidates for

vaccine formulations has been described in many publications.

In some of these publications, research on some small peptide

immunostimulants used for vaccine adjuvant strategies has also

been reported （2, 8, 36）.

In our previous study, we isolated and puri?ed a novel bursa

pentapeptide, BP5, which was capable of enhancing antigen-

speci?c humoral and cell-mediated immune responses in mice

（19）. In the present study, we found that a simple mixture of

inactivated H9N2 AIVs and BP5 also enhanced humoral and

cell-mediated immune responses in chickens. When coinjected

with the model antigen （an inactivated avian in?uenza virus

[AIV], A/Duck/Jiangsu/NJ08/05 [AIV H9N2 subtype]）, BP5

FIG. 3. BP5 signi?cantly stimulates chicken peripheral blood lym-

phocyte proliferation. Chickens were immunized two times, and

chicken peripheral blood lymphocytes were collected on day 28

postimmunization. Proliferative response was evaluated byMTT assay.

Data are the means SD of results from four separate experiments.

, P 0.05, compared to results with PBS alone; #, P 0.05, com-

pared to results with AIVs alone; †, P 0.05, compared to chickens

immunized with the commercial H9N2 AIV vaccine.

TABLE 2. Flow cytometric analysis of CD3 T cells and their

subsets CD3 CD4 and CD3 CD8 T cells from the

peripheral blood lymphocytes of immunized chickens

a

Treatment

% of peripheral blood lymphocytes of type:

CD3 CD3 CD4 CD3 CD8

PBS 35.15 2.14 14.36 1.89 9.22 1.51

AIVs 45.99 1.23 20.87 2.13 13.68 2.12

AIVs 25 mg/kg BP5 48.78 2.86 20.54 1.38 16.87 1.89

AIVs 5 mg/kg BP5 58.82 2.48* 26.89 1.56* 22.67 1.78*†

AIVs 1 mg/kg BP5 61.46 1.61**† 30.44 2.24** 25.01 1.53**†

H9N2 AIV vaccine 56.87 2.31* 28.57 1.79** 16.51 1.35

a

Chickens were sacri?ced on day 28 after ?rst immunization, and the periph-

eral blood lymphocytes were collected for immunophenotyping. The data pre-

sented are means SD of results from four replicates. *, P 0.05, and **, P

0.01, compared with chickens immunized with the inactivated AIVs alone. †, P

0.05, compared with chickens immunized with the commercial H9N2 AIV vac-

cine.

TABLE 3. Detection of AIV copies in the lungs of H9N2

AIV-challenged chickens by a SYBR green 1 real-time PCRa

Group

No. of lung viral copies （log 10）/ml

PBS on day postchallenge:

135

PBS 8.6 0.015 7.2 0.036 4.5 0.045

AIVs 7.1 0.023 5.5 0.024 3.1 0.054

AIVs 25 mg/kg BP5 5.7 0.034* 4.4 0.028* 1.9 0.027*

AIVs 5 mg/kg BP5 5.4 0.042* 4.0 0.064* 1.5 0.039*

AIVs 1 mg/kg BP5 5.0 0.041**† 3.1 0.055**† 0.9 0.034**†

H9N2 AIV Vaccine 6.0 0.031* 4.3 0.034* 2.2 0.035*

a

Lung samples from individual chickens in each group were collected on days

1, 3, and 5 postchallenge. Each lung sample was diluted to 1 ml with PBS. The

titers are presented as numbers of copies per ml PBS. The data presented are

means SD of results from ?ve replicates. *, P 0.05, and **, P 0.01,

compared to chickens immunized with AIVs alone. †, P 0.05, compared with

chickens immunized with the commercial H9N2 AIV vaccine.

1500 LI ET AL. CLIN.VACCINE IMMUNOL.

 on April 26, 2012 by Huazhong Agricultural University http://cvi.asm.org/ Downloaded from induced higher levels of antigen-speci?c hemagglutination in-

hibition （HI） antibody titers and antigen-speci?c HA antibody

（IgG） titers in chickens than were induced in chickens immu-

nized with inactivated avian in?uenza virus alone. Further-

more, chickens coadministered inactivated AIVs and proper

concentrations of BP5 （used as an adjuvant） produced signif-

icantly higher HI and IgG antibody titers than chickens immu-

nized with the commercial H9N2 AIV vaccine （prepared with

oil/water as an adjuvant）. In some literature, it has been re-

ported that a single administration of commercial H9N2 AIV

vaccine in oil emulsion induced higher HI antibody titers

（about 9 log2） 3 weeks after vaccination than the control （16）,

whereas in other literature, it has been reported that oil adju-

vant H9N2 AIV vaccine produced HI antibody titers that were

less than 6 log2 2 weeks after the ?rst vaccination, less than 7.0

log2 3 weeks after the ?rst vaccination, and less than 8.0 log2 3

weeks after the second vaccination （17）. It is well known that

various factors, like source of erythrocytes, type of diluent,

incubation temperature, and incubation period, affect hemag-

glutination activity, and thereby, they affect the HI titers （12）.

In view of this, the data for HI antibody titers obtained from

this study are generally consistent with the data reported by

Lee et al. （17）. Although the HI antibody titers induced by the

commercial AIV vaccine and by BP5 adjuvant-inactivated

AIVs were not very high in this study, BP5 adjuvant-inacti-

vated AIVs induced higher HI antibody titers than oil adjuvant

commercial AIV vaccine. This suggested that BP5 has an ef-

fective adjuvant activity in vaccines that enhances antigen-

speci?c humoral immune responses.

In addition to humoral responses, cellular immunity plays an

important role in ?ghting in?uenza virus infections （14）. In this

study, cell-mediated immunity was evaluated in vaccinated

chickens through cytokine analysis and in vitro proliferation

assay of peripheral blood splenocytes pre- and postimmuniza-

tion. Currently, special attention is being given to adjuvants

capable of ef?ciently promoting a Th1-type immune response,

which is considered the best correlate of a protective immune

response to infections （32）. However, the most powerful Th1-

promoting adjuvants exhibit some toxicity, which limits their

clinical use （27）. The most remarkable ?nding reported in the

present study is the demonstration that BP5, coadministered

with inactivated AIVs, represents an unexpectedly powerful

adjuvant, not only inducing the production of Th1-type cyto-

kines （IL-2 and IFN-） but also inducing the production of

Th2-type cytokines （IL-4）. Moreover, in vivo/ex vivo, using

MTT incorporation to measure cell proliferation and ?ow cy-

tometric analysis to measure immunophenotyping of T lym-

phocytes, signi?cant increases in peripheral blood lymphocyte

proliferation and in the sizes of CD3 T cell populations,

including CD3 CD4 and CD3 CD8 T cell populations,

were found in chickens coadministered inactivated AIVs and

BP5. In contrast, although the levels of cytokines in sera

and the levels of peripheral blood lymphocyte proliferation

and CD3 T cell populations were increased in chickens im-

munized with a commercial, inactivated AIV vaccine （pre-

pared with oil/water as an adjuvant）, levels of only Th1-type

cytokines increased, and the CD8 T cells were only moder-

ay affected. These results indicate that BP5 has the potential

to affect cell-mediated responses and balance Th1- and Th2-

type immune responses when used as an adjuvant.

To further evaluate the in?uence of BP5 as an adjuvant on

the immunity protection provided by inactivated AIVs against

avian in?uenza virus infection, chickens were challenged intra-

nasally with avian in?uenza H9N2 virus strain JS-1 （A/Chicken/

Jiangsu/JS-1/2002） on day 28 after they had been coimmu-

nized with inactivated AIVs and BP5. At 2 days postchallenge,

the nonvaccinated chickens that received the challenge virus

were mildly depressed. No other clinical signs were observed in

that group or any of the other groups, which is typical of

low-pathogenicity AIVs in chickens （15, 33）. At 5 days post-

challenge, only the nonvaccinated challenged group had mild,

grossly detectable lesions in both the respiratory and gastroin-

testinal tract. As JS-1 H9N2 virus is a low-pathogenicity avian

in?uenza virus and all challenged chickens survived the infec-

tions, we used SYBR green 1-based real-time PCR to assess

the extent of virus infection, monitoring the protection level of

inactivated AIVs after they were coadministered with BP5.We

detected the challenge virus in the lungs of the challenged

chickens on days 1, 3, and 5. Our data indicated that viral

replication （viral shedding） occurred and that virus shedding

could be more ef?ciently blocked or reduced after a homolo-

gous vaccine was coadministered with BP5, which was used to

vaccinate chickens against the challenge virus. This result sug-

gests that BP5 has the potential to be used in vaccine formu-

lations to provide improved protection against H9N2 AIV

infection in poultry.

Several small peptides have been synthesized in an effort to

discover an idealized peptide sequence with signi?cant immu-

nological adjuvant activity （7, 8）. Our previous study revealed

that B lymphocyte proliferation induced by BP5 is mediated by

reactive oxygen species generated from thiol auto-oxidation of

Cys in BP5 （19）. We presume that Cys plays an important role

in the immune functions of BP5. Thus, analogs of BP5, such as

Gly-Lys-Asp-Val-Tyr, Ala-Lys-Asp-Val-Tyr, and Glu-Lys-Asp-

Val-Tyr, were also synthesized and used to evaluate their im-

mune activities in mice and chickens. In the assays, no signif-

icant immune adjuvant activities of these peptides were

detected （data not shown）. This suggests that the speci?c im-

mune inducer properties of BP5 are associated with its special

amino acid sequence. Further research on the relationship

between the structure and the immune activity of BP5 will

contribute new insights into the mechanisms of adjuvant activ-

ity and may lead to the development of a practical application

in vaccine design. Further studies are also needed to further

compare the effects of BP5 and other adjuvants.

In summary, we demonstrated that BP5 enhanced the avian

in?uenza virus-speci?c cell-mediated and humoral immune re-

sponses induced by inactivated AIVs. Furthermore, intramus-

cular immunization with a mixture of inactivated AIVs and

BP5 enhanced protection against a homologous avian in?u-

enza virus challenge by reducing viral replication in chicken

lungs. This study indicates that BP5 possesses adjuvant activi-

ties and that it may be used as a new experimental reagent for

immuno-adjuvant uses.

ACKNOWLEDGMENT

The present study was supported by a grant from the National

Agriculture Special Research Project （grant 200803020）.

VOL. 18, 2011 BURSOPENTINE （BP5） AS A NOVEL IMMUNOADJUVANT 1501

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