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Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant a
a
a
a
a
b
c
Mei Zheng , Fen Liu , Yiqing Shen , Shilei Wang , Wenting Xu , Fang Fang , Bing Sun , d
ab
Zhenyuan Xie & Ze Chen a
Shanghai Institute of Biological Products, Shanghai 200052, China;
b
College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China;
c
Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China; d
Click for updates
Xie Tu Community Medical Service Center, Xuhui District, Shanghai, China, 200032 Accepted author version posted online: 21 Jan 2015.
To cite this article: Mei Zheng, Fen Liu, Yiqing Shen, Shilei Wang, Wenting Xu, Fang Fang, Bing Sun, Zhenyuan Xie & Ze Chen (2015): Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant, Human Vaccines & Immunotherapeutics, DOI: 10.4161/21645515.2014.995056 To link to this article: http://dx.doi.org/10.4161/21645515.2014.995056
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant Mei Zheng1, Fen Liu1, Yiqing Shen1, Shilei Wang1, Wenting Xu1, Fang Fang2, Bing
Shanghai Institute of Biological Products, Shanghai 200052, China; 2 College of Life
cr
Sciences, Hunan Normal University, Changsha 410081, Hunan, China; 3 Institute
us
Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of
an
Sciences, Shanghai 200025, China; 4 Xie Tu Community Medical Service Center, Xuhui District, Shanghai, China, 200032 *
M
co-corresponding authors:
Zhenyuan Xie: Xie Tu Community Medical Service Center, Xuhui District, Shanghai,
ed
China, 200032. Email : [email protected]. Ze Chen: Shanghai Institute of Biological Products, Shanghai 200052, China. Email: [email protected];
pt
[email protected];
Phone/Fax:
(00)
86-(0)
ce
21-62826658.
Abstract: The nucleoprotein (NP) of influenza viruses is highly conserved and therefore has become one of the major targets of current universal influenza vaccine
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
1
ip t
Sun3, Zhenyuan Xie4, *, and Ze Chen1,2,*
(UIV) studies. In this study, the recombinant nucleoprotein (NP) of the A/PR/8/34 (H1N1) influenza virus strain was expressed using an Escherichia coli (E. coli) expression system and then purified as a candidate UIV. The NP protein was administered intranasally or intraperitoneally twice at 3-week intervals to female 1
BALB/c mice in combination with C48/80 adjuvant. Then, the mice were challenged with homologous or heterologous influenza viruses at a lethal dose three weeks after the last immunization. The results showed that the serum IgG titers of all of the mice
ip t
immunized with NP reached a higher level and the protection provided by NP vaccine against the homologous virus depended on the administered dosage and adjuvant. In
cr
us
provide good cross-protection against heterologous H9N2 avian influenza viruses. This study indicated that NP as a candidate antigen of UIV immunized intranasally
an
could effectively induce mucosal and cell-mediated immunity, with the potential to control epidemics caused by the appearance of new emerging influenza viruses.
ce
pt
ed
Compound 48/80
M
Keywords: Influenza; Intranasal immunization; Mucosal adjuvant; nucleoprotein;
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
addition, immunization with 100mg NP in combination with C48/80 adjuvant could
2
1. Introduction
Influenza is an acute respiratory disease caused by infection of the host respiratory
ip t
tract by influenza virus, and vaccination is one of the most effective means against
cr
and neuraminidase (NA), are the major antigen of influenza vaccines. However, HA
us
and NA are prone to antigenic drift, and it is difficult to ensure antigens of vaccine
an
strains match those in the strains circulating in the next season. Therefore, developing an effective and long-lasting universal influenza vaccine (UIV) has become a research
M
priority at present. Once a pandemic influenza virus breaks out, it is a simply and rapid strategy to express and purify the antigen protein based on the expression
ed
system of bacteria, yeasts or baculoviruses.
pt
The NP protein of influenza viruses is highly conserved and therefore has become one of the major targets of current UIV studies. Influenza virus nucleoprotein (NP) is
ce
type-specific, and is one of the main determinants of species specificity.28 It has highly conserved sequences and low mutation rate during the evolution of viruses. NP
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
influenza virus infections. Currently, two surface glycoproteins, hemaglutinin (HA)
proteins of influenza viruses of the same type have over 90% amino acid homology.1 NP participates in the transcription and duplication of virus genome, and affects the host specificity and virulence of viruses.29 Following infection of influenza virus, NP is the main antigen recognized by host cytotoxic T lymphocytes (CTL).1 Through recognizing NP antigen peptides presented by MHC-I molecules on the surface of 3
virus infected cells, CTL then kills virus infected cells and thereby eliminate the virus.2 Cross-reactive CTL aimed at NP protein play an important role in controlling influenza virus infection.1,2 There are many successful reports in the animal model
ip t
studies about NP-based influenza vaccines. 3,4,25,30 Diverse vaccines with NP as the target antigen have been designed accordingly, including the diversely-designed DNA
cr
us
and prokaryotic expression, as well as the viral vector vaccines,3, 4 which can induce certain heterosubtypic immune response. Moreover, recombinant protein vaccines
an
have excellent safety, and can induce adequate antibody levels in a population
development at present.31
M
vaccination, and therefore have become an important direction of influenza vaccine
ed
However, if a new pandemic influenza virus breaks out, vaccination with a recombinant protein vaccine seems to be insufficient. Therefore, to further improve
pt
effectiveness of UIV, researchers tried to achieve the desired goals through combined
ce
use of multiple target antigens. In addition, recombinant protein vaccines require adjuvants to improve their ability of eliciting an effective immune response. Using a safe adjuvant with a vaccine will increase immune response and can lower dose of
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
vaccines, the genetically engineered vaccines of insect baculovirus system expression
antigen required. Some adjuvants have been found to have the potential to enhance the immunogenicity of UIVs. For example, some mucosal adjuvants such as chitosan, lipid and E. coli thermosensitive toxin (LT) can induce balanced Th1/Th2 immune response by intranasal (i.n.) immunization in combination with vaccines.5 Compound 48/80 (C48/80) is a polymer produced by the mixture of dimer, trimer and tetramer of 4
concentrate of p-methoxyphenethyl-methylamine and formaldehyde, and plays a role of activating the mast cell (MC), so it is named MC activator.6 C48/80 has been recognized and understood as a MC activator for a long time, but in recent studies, it
ip t
is found to be a potential mucosal adjuvant. Just like other mucosal adjuvants, C48/80 can also induce the effective sIgA at the mucosa. In addition, C48/80 has also been
cr
us
In this study, the NP protein was expressed using E. coli expression system and then purified as a subunit vaccine, and was immunized intranasally to mice in combination
an
with C48/80 adjuvant. It was found that NP, as a candidate vaccine, could protect mice against the influenza virus challenge, and that C48/80 adjuvant could
M
significantly enhance the protective effect of the NP vaccine.
ed
2. Results
pt
2.1 Intranasal administration of NP protein with C48/80 protected mice from lethal
ce
H1N1 virus challenge
To investigate whether intranasal administration of NP protein could protect mice against influenza virus infection, the Plasmid pET28a/NP was transformed into
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
confirmed as a nontoxic nasal adjuvant.7
Escherichia coli (E.coli) BL21 (DE3) bacteria, and then expressed and purified the NP protein as a subunit vaccine as described by our previous study,3 followed by immunized intranasally to mice with C48/80 adjuvant. One hundred fourty-three mice were divided randomly into 11 groups of 13 each (Group A-K, Table 1 and S1). The mice (Groups A-H) were immunized by i.n. administration, and the mice (Groups I 5
and J) were immunized intraperitoneally (i.p.) with 100μg NP in combination with or without 60μg C48/80 adjuvant. Group K was the unimmunized control. The mice intranasally immunized with 100, 30, 10μg NP alone (Group B, D and F), or in
ip t
combination with 60μg C48/80 adjuvant (Group A, C and E). In addition, the mice intranasally immunized with C48/80 alone (Group G) were the adjuvant control, the
cr
us
positive control. Each mouse was i.n. or i.p. administered twice at an interval of 3 weeks with different amounts of NP with or without adjuvants. Three weeks after the
an
last immunization, the mice were challenged intranasally with 10LD50 A/PR/8/34 (H1N1) virus suspension, and bronchoalveolar washes of 3 mice in each group were
M
used for the analyses of virus loads in the lungs 3 days postinfection, while the rest of
ed
them were monitored to observe the survival rates and weight loss of the mice. The results showed that 100µg NP protein with C48/80 adjuvant could enhance the
pt
immune effect after immunized mice. As shown in table 1, 0% (0/10), 20% (2/10) and
ce
40% (4/10) survival rates were obtained when challenges after immunized with 10mg, 30mg, and 100mg NP protein alone (Group F, D and B), if the mice were immunizied with the same dosage of NP plus C48/80 adjuvant, higher levels of protection were
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
mice intranasally immunized with 100μg NP plus 1μg CTB★ (Group H) were the
achieved [20% (2/10), 40% (4/10) and 100% (10/10)] ( Group E, C and A). The mice injected intraperitoneally with 100mg NP with adjuvant could not be effectively protected (Table1, Fig.1A). In Figure 1, after intranasal immunization, the survival rate of mice in Group A, B, C and H had significant difference compared with unimmunized group (p < 0.05). And the survival rate of mice in Group A, C and H 6
also had significant difference compared with the same doses of group without adjuvant (p < 0.05). As shown in Table1, the group immunized with NP alone had a stronger protective effect along with the increased NP dose, and at the same time, the
ip t
combination with C48/80 adjuvant could effectively enhanced protective immunity induced by NP.
cr
us
survival rates in mice. As shown in the figure1B, the body weight loss was slightly slower and the symptoms was mildest in the mice immunized with 100mg NP plus
an
60mg C48/80 and 1mg CTB★ adjuvant respectively (Fig.1B and 1C). The results also showed that the mice in Group B and Group D lost body weight slower, and they
M
quickly recovered to the normal weight, while the rest of them in other groups began
ed
to recover their body weights 9 days after challenge. As shown in Table 1, three days after challenge, the viral loads in the trachea/lung
pt
tissues from the mice that were immunized intranasally with 100mg NP plus 60mg
ce
C48/80 and 1mg CTB★ adjuvant were significantly lower than that from the unimmunized control group (p
Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant a
a
a
a
a
b
c
Mei Zheng , Fen Liu , Yiqing Shen , Shilei Wang , Wenting Xu , Fang Fang , Bing Sun , d
ab
Zhenyuan Xie & Ze Chen a
Shanghai Institute of Biological Products, Shanghai 200052, China;
b
College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China;
c
Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China; d
Click for updates
Xie Tu Community Medical Service Center, Xuhui District, Shanghai, China, 200032 Accepted author version posted online: 21 Jan 2015.
To cite this article: Mei Zheng, Fen Liu, Yiqing Shen, Shilei Wang, Wenting Xu, Fang Fang, Bing Sun, Zhenyuan Xie & Ze Chen (2015): Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant, Human Vaccines & Immunotherapeutics, DOI: 10.4161/21645515.2014.995056 To link to this article: http://dx.doi.org/10.4161/21645515.2014.995056
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant Mei Zheng1, Fen Liu1, Yiqing Shen1, Shilei Wang1, Wenting Xu1, Fang Fang2, Bing
Shanghai Institute of Biological Products, Shanghai 200052, China; 2 College of Life
cr
Sciences, Hunan Normal University, Changsha 410081, Hunan, China; 3 Institute
us
Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of
an
Sciences, Shanghai 200025, China; 4 Xie Tu Community Medical Service Center, Xuhui District, Shanghai, China, 200032 *
M
co-corresponding authors:
Zhenyuan Xie: Xie Tu Community Medical Service Center, Xuhui District, Shanghai,
ed
China, 200032. Email : [email protected]. Ze Chen: Shanghai Institute of Biological Products, Shanghai 200052, China. Email: [email protected];
pt
[email protected];
Phone/Fax:
(00)
86-(0)
ce
21-62826658.
Abstract: The nucleoprotein (NP) of influenza viruses is highly conserved and therefore has become one of the major targets of current universal influenza vaccine
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
1
ip t
Sun3, Zhenyuan Xie4, *, and Ze Chen1,2,*
(UIV) studies. In this study, the recombinant nucleoprotein (NP) of the A/PR/8/34 (H1N1) influenza virus strain was expressed using an Escherichia coli (E. coli) expression system and then purified as a candidate UIV. The NP protein was administered intranasally or intraperitoneally twice at 3-week intervals to female 1
BALB/c mice in combination with C48/80 adjuvant. Then, the mice were challenged with homologous or heterologous influenza viruses at a lethal dose three weeks after the last immunization. The results showed that the serum IgG titers of all of the mice
ip t
immunized with NP reached a higher level and the protection provided by NP vaccine against the homologous virus depended on the administered dosage and adjuvant. In
cr
us
provide good cross-protection against heterologous H9N2 avian influenza viruses. This study indicated that NP as a candidate antigen of UIV immunized intranasally
an
could effectively induce mucosal and cell-mediated immunity, with the potential to control epidemics caused by the appearance of new emerging influenza viruses.
ce
pt
ed
Compound 48/80
M
Keywords: Influenza; Intranasal immunization; Mucosal adjuvant; nucleoprotein;
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
addition, immunization with 100mg NP in combination with C48/80 adjuvant could
2
1. Introduction
Influenza is an acute respiratory disease caused by infection of the host respiratory
ip t
tract by influenza virus, and vaccination is one of the most effective means against
cr
and neuraminidase (NA), are the major antigen of influenza vaccines. However, HA
us
and NA are prone to antigenic drift, and it is difficult to ensure antigens of vaccine
an
strains match those in the strains circulating in the next season. Therefore, developing an effective and long-lasting universal influenza vaccine (UIV) has become a research
M
priority at present. Once a pandemic influenza virus breaks out, it is a simply and rapid strategy to express and purify the antigen protein based on the expression
ed
system of bacteria, yeasts or baculoviruses.
pt
The NP protein of influenza viruses is highly conserved and therefore has become one of the major targets of current UIV studies. Influenza virus nucleoprotein (NP) is
ce
type-specific, and is one of the main determinants of species specificity.28 It has highly conserved sequences and low mutation rate during the evolution of viruses. NP
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
influenza virus infections. Currently, two surface glycoproteins, hemaglutinin (HA)
proteins of influenza viruses of the same type have over 90% amino acid homology.1 NP participates in the transcription and duplication of virus genome, and affects the host specificity and virulence of viruses.29 Following infection of influenza virus, NP is the main antigen recognized by host cytotoxic T lymphocytes (CTL).1 Through recognizing NP antigen peptides presented by MHC-I molecules on the surface of 3
virus infected cells, CTL then kills virus infected cells and thereby eliminate the virus.2 Cross-reactive CTL aimed at NP protein play an important role in controlling influenza virus infection.1,2 There are many successful reports in the animal model
ip t
studies about NP-based influenza vaccines. 3,4,25,30 Diverse vaccines with NP as the target antigen have been designed accordingly, including the diversely-designed DNA
cr
us
and prokaryotic expression, as well as the viral vector vaccines,3, 4 which can induce certain heterosubtypic immune response. Moreover, recombinant protein vaccines
an
have excellent safety, and can induce adequate antibody levels in a population
development at present.31
M
vaccination, and therefore have become an important direction of influenza vaccine
ed
However, if a new pandemic influenza virus breaks out, vaccination with a recombinant protein vaccine seems to be insufficient. Therefore, to further improve
pt
effectiveness of UIV, researchers tried to achieve the desired goals through combined
ce
use of multiple target antigens. In addition, recombinant protein vaccines require adjuvants to improve their ability of eliciting an effective immune response. Using a safe adjuvant with a vaccine will increase immune response and can lower dose of
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
vaccines, the genetically engineered vaccines of insect baculovirus system expression
antigen required. Some adjuvants have been found to have the potential to enhance the immunogenicity of UIVs. For example, some mucosal adjuvants such as chitosan, lipid and E. coli thermosensitive toxin (LT) can induce balanced Th1/Th2 immune response by intranasal (i.n.) immunization in combination with vaccines.5 Compound 48/80 (C48/80) is a polymer produced by the mixture of dimer, trimer and tetramer of 4
concentrate of p-methoxyphenethyl-methylamine and formaldehyde, and plays a role of activating the mast cell (MC), so it is named MC activator.6 C48/80 has been recognized and understood as a MC activator for a long time, but in recent studies, it
ip t
is found to be a potential mucosal adjuvant. Just like other mucosal adjuvants, C48/80 can also induce the effective sIgA at the mucosa. In addition, C48/80 has also been
cr
us
In this study, the NP protein was expressed using E. coli expression system and then purified as a subunit vaccine, and was immunized intranasally to mice in combination
an
with C48/80 adjuvant. It was found that NP, as a candidate vaccine, could protect mice against the influenza virus challenge, and that C48/80 adjuvant could
M
significantly enhance the protective effect of the NP vaccine.
ed
2. Results
pt
2.1 Intranasal administration of NP protein with C48/80 protected mice from lethal
ce
H1N1 virus challenge
To investigate whether intranasal administration of NP protein could protect mice against influenza virus infection, the Plasmid pET28a/NP was transformed into
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
confirmed as a nontoxic nasal adjuvant.7
Escherichia coli (E.coli) BL21 (DE3) bacteria, and then expressed and purified the NP protein as a subunit vaccine as described by our previous study,3 followed by immunized intranasally to mice with C48/80 adjuvant. One hundred fourty-three mice were divided randomly into 11 groups of 13 each (Group A-K, Table 1 and S1). The mice (Groups A-H) were immunized by i.n. administration, and the mice (Groups I 5
and J) were immunized intraperitoneally (i.p.) with 100μg NP in combination with or without 60μg C48/80 adjuvant. Group K was the unimmunized control. The mice intranasally immunized with 100, 30, 10μg NP alone (Group B, D and F), or in
ip t
combination with 60μg C48/80 adjuvant (Group A, C and E). In addition, the mice intranasally immunized with C48/80 alone (Group G) were the adjuvant control, the
cr
us
positive control. Each mouse was i.n. or i.p. administered twice at an interval of 3 weeks with different amounts of NP with or without adjuvants. Three weeks after the
an
last immunization, the mice were challenged intranasally with 10LD50 A/PR/8/34 (H1N1) virus suspension, and bronchoalveolar washes of 3 mice in each group were
M
used for the analyses of virus loads in the lungs 3 days postinfection, while the rest of
ed
them were monitored to observe the survival rates and weight loss of the mice. The results showed that 100µg NP protein with C48/80 adjuvant could enhance the
pt
immune effect after immunized mice. As shown in table 1, 0% (0/10), 20% (2/10) and
ce
40% (4/10) survival rates were obtained when challenges after immunized with 10mg, 30mg, and 100mg NP protein alone (Group F, D and B), if the mice were immunizied with the same dosage of NP plus C48/80 adjuvant, higher levels of protection were
Ac
Downloaded by [McGill University Library] at 19:28 08 February 2015
mice intranasally immunized with 100μg NP plus 1μg CTB★ (Group H) were the
achieved [20% (2/10), 40% (4/10) and 100% (10/10)] ( Group E, C and A). The mice injected intraperitoneally with 100mg NP with adjuvant could not be effectively protected (Table1, Fig.1A). In Figure 1, after intranasal immunization, the survival rate of mice in Group A, B, C and H had significant difference compared with unimmunized group (p < 0.05). And the survival rate of mice in Group A, C and H 6
also had significant difference compared with the same doses of group without adjuvant (p < 0.05). As shown in Table1, the group immunized with NP alone had a stronger protective effect along with the increased NP dose, and at the same time, the
ip t
combination with C48/80 adjuvant could effectively enhanced protective immunity induced by NP.
cr
us
survival rates in mice. As shown in the figure1B, the body weight loss was slightly slower and the symptoms was mildest in the mice immunized with 100mg NP plus
an
60mg C48/80 and 1mg CTB★ adjuvant respectively (Fig.1B and 1C). The results also showed that the mice in Group B and Group D lost body weight slower, and they
M
quickly recovered to the normal weight, while the rest of them in other groups began
ed
to recover their body weights 9 days after challenge. As shown in Table 1, three days after challenge, the viral loads in the trachea/lung
pt
tissues from the mice that were immunized intranasally with 100mg NP plus 60mg
ce
C48/80 and 1mg CTB★ adjuvant were significantly lower than that from the unimmunized control group (p
