JGV Papers in Press. Published April 23, 2015 as doi:10.1099/vir.0.000160

Journal of General Virology Genotype-specific neutralization determinant in envelope protein: implications for the improvement of Japanese Encephalitis vaccine --Manuscript Draft-Manuscript Number:

VIR-D-15-00192R1

Full Title:

Genotype-specific neutralization determinant in envelope protein: implications for the improvement of Japanese Encephalitis vaccine

Short Title:

JEV genotype-specific neutralization determinants

Article Type:

Standard

Section/Category:

Animal - Positive-strand RNA Viruses

Corresponding Author:

Cheng-Feng Qin Beijing Institute of Microbiology and Epidemiology Beijing, CHINA

First Author:

Qing Ye

Order of Authors:

Qing Ye Yan-Peng Xu Yu Zhang Xiao-Feng Li Hong-Jiang Wang Zhong-Yu Liu Shi-Hua Li Long Liu Hui Zhao Qing-Gong Nian Yong-Qiang Deng E-De Qin Cheng-Feng Qin

Abstract:

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in children in Asia and is expanding its geographic range to larger areas in Asia and Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in its geographically affected areas. Especially, the emergence of genotype I (GI) JEV has displaced genotype III (GIII) as dominant circulating strains in many Asian regions. However, all approved vaccine products are derived from GIII strains. In the present study, bioinformatic analysis revealed GI and GIII JEV strains shared two distinct amino acid residues within envelope (E) protein (E222 and E327). By using reverse genetic approaches, A222S and S327T mutations were demonstrated to decrease the liveattenuated vaccine (LAV) SA14-14-2 induced neutralizing antibody in human, without altering viral replication. Then, A222S or S327T mutations were rationally engineered into the infectious clone of SA14-14-2, respectively, and the resulting mutant strains remained the same genetic stability and attenuation characteristics as their parent strain. More importantly, immunization of mice with LAV-A222S or LAV-S327T elicited increased neutralizing antibodies against GI strains. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants, and are critical in determining the protective efficacy of live JE vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the rational design of next generation of live-attenuated JE vaccine capable of providing broad protection against all JEV strains belonging to different genotypes.

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Title Page

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Genotype-specific neutralization determinant in envelope protein: implications

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for the improvement of Japanese Encephalitis vaccine

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Qing Yea†, Yan-Peng Xua†, Yu Zhang a, Xiao-Feng Lia,b, Hong-Jiang Wanga,

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Zhong-Yu Liua, Shi-Hua Lia, Long Liua,c, Hui Zhaoa,b, Qing-Gong Niana, Yong-Qiang

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Denga, E-De Qin a, Cheng-Feng Qin a,b,c*

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a

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Beijing 100071, China

Department of Virology, Beijing Institute of Microbiology and Epidemiology,

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b

State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, China

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c

Graduate School, Anhui Medical University, Hefei 230032, Chin

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†These authors contribute equally to this work.

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*Corresponding author. Mailing address: Department of Virology, Beijing Institute of

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Microbiology and Epidemiology, Beijing 100071, China, Email: [email protected]

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Content category: Animal RNA viruses

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Running title: JEV genotype-specific neutralization determinants

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Number of words in the summary: 229

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Total number of words in the main text: 3455

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Number of Figures: 5

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1 2

ABSTRACT

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Japanese encephalitis (JE) remains the leading cause of viral encephalitis in

4

children in Asia and is expanding its geographic range to larger areas in Asia and

5

Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in its

6

geographically affected areas. Especially, the emergence of genotype I (GI) JEV has

7

displaced genotype III (GIII) as dominant circulating strains in many Asian regions.

8

However, all approved vaccine products are derived from GIII strains. In the present

9

study, bioinformatic analysis revealed GI and GIII JEV strains shared two distinct

10

amino acid residues within envelope (E) protein (E222 and E327). By using reverse

11

genetic approaches, A222S and S327T mutations were demonstrated to decrease the

12

live-attenuated vaccine (LAV) SA14-14-2 induced neutralizing antibody in human,

13

without altering viral replication. Then, A222S or S327T mutations were rationally

14

engineered into the infectious clone of SA14-14-2, respectively, and the resulting

15

mutant strains remained the same genetic stability and attenuation characteristics as

16

their parent strain. More importantly, immunization of mice with LAV-A222S or

17

LAV-S327T elicited increased neutralizing antibodies against GI strains. Together,

18

these results demonstrated that E222 and E327 are potential genotype-related

19

neutralization determinants, and are critical in determining the protective efficacy of

20

live JE vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the

21

rational design of next generation of live-attenuated JE vaccine capable of providing

22

broad protection against all JEV strains belonging to different genotypes.

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Key words: Japanese encephalitis virus; Genotype; Live attenuated vaccine; Neutralization

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INTRODUCTION

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Japanese encephalitis (JE), a typical mosquito-borne viral disease, is one of the most

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important causes of viral encephalitis resulting in about 68,000 cases and

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10,000-15,000 deaths worldwide annually (Solomon et al., 2003; van den Hurk et al.,

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2009). Japanese encephalitis virus (JEV) is transmitted by Culex mosquitos between

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vertebrate hosts, particularly pigs and birds, and humans are occasional dead-end

8

hosts. Clinical manifestation of JEV infection ranges from undifferentiated febrile

9

illness and aseptic meningitis to acute encephalitis, and death. About half of survivors

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have severe neurological sequelae (Solomon et al., 2000). The geographical range of

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JE is expanding to larger areas in Asia and Australia during the last decade (Hanna et

12

al., 1996; Mackenzie et al., 2004). To date, JE affects more than 25 countries, where

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~60% of the global population lives at risk of JEV infection.

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JEV is a member of the family Flaviviridae, genus Flavivirus, together with other

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important human pathogens including dengue virus (DENV), West Nile virus (WNV),

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yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV). JEV has an

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approximately 11 kb positive-sense, single-stranded RNA genome, which contains a

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single open reading frame that encodes three structural proteins (capsid, premembrane,

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and envelope) and seven nonstructural proteins (NS1 to NS5), flanked by the 5’ and 3’

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untranslated regions (UTRs). Based on the envelope (E) gene sequence or the

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complete genome, JEV can be further divided into five genotypes (GI to GV) (Uchil

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& Satchidanandam, 2001), and each genotype has a specific geographic distribution

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pattern. Currently, GI and GIII strains represent the most prevalent genotypes in many

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epidemic countries. In the first half of 20th century, the majority of JEV isolates from

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human belong to GIII; however, since the last two decades, GI virus has gradually

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replaced GIII as the dominant circulating strains in many areas of Asia including

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China, Vietnam, Japan, India and Thailand (Ma et al., 2003; Nga, 2004; Nitatpattana

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et al., 2008; Pan et al., 2011; Parida et al., 2006; Sarkar et al., 2012). The mechanism

29

underlying the genotype displacement as well as the impact of the genotype shift on

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transmission/outbreak control strategies remains largely unknown (Han et al., 2014; 3

1

Schuh et al., 2013; Schuh et al., 2014).

2

Vaccination has been proven the most effective method to prevent and control JE,

3

and the incidence has decreased significantly in many Asian countries. Currently,

4

several inactivated vaccines and live attenuated vaccines have been licensed for

5

clinical use (Beasley et al., 2008; Halstead & Thomas, 2010). The inactivated

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vaccines are cultivated in mouse brain, primary hamster kidney cells or Vero cells.

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The live attenuated vaccine SA14-14-2 was first licensed in 1989 in China, and now

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has been widely used in JEV-endemic countries in Asia including China, South Korea,

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Thailand, India, Nepal and Sri Lanka (Gatchalian et al., 2008; Yu, 2010). More than

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300 million children have been immunized with LAV SA14-14-2, and the safety and

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efficacy profile has been well established (Bista et al., 2001; Hennessy et al., 1996;

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Kumar et al., 2009; Liu et al., 1997; Tsai et al., 1998). Very recently, a recombinant

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chimeric LAV based on the yellow fever vaccine strain (17D) backbone carrying the

14

prM and E gene of JEV SA14-14-2 (JE-CV) has been licensed in multiple countries

15

including Australia, Thailand, Malaysia and Philippines (Bonaparte et al., 2014;

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Feroldi et al., 2014; Monath et al., 2015). However, all the licensed JE vaccines,

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including SA14-14-2, are derived from GIII viruses. Previous clinical trials with GIII

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vaccines in areas where heterologous genotypes are circulating have showed

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effectiveness. It has been also expected that immunity induced by GIII vaccines

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would provide cross protection against the infection of other JEV genotypes. However,

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various immunological assays with antisera or monoclonal antibodies have

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demonstrated the existence of antigenic differences among genotypes of JEV strains

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(Hasegawa et al., 1994; Hasegawa et al., 1995; Kimura-Kuroda & Yasui, 1983;

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Kobayashi et al., 1985; Wills et al., 1993). Data from inactivated GIII vaccines

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demonstrated reduced neutralization capability against heterologous GI viruses

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(Beasley et al., 2004; Erra et al., 2013; Fan et al., 2012; Ferguson et al., 2008; Kurane

27

& Takasaki, 2000). Mice immunized with SA14-14-2 showed decreased

28

neutralization antibody titers against GI viruses (Liu et al., 2011). Recently, we also

29

demonstrated that the LAV vaccination induced reduced levels of neutralizing

30

antibody against GI viruses in human (Ye et al., 2014). Especially, several groups 4

1

reported JE cases in individuals who have been vaccinated with LAV SA14-14-2 (Hu

2

et al., 2013; Sarkar et al., 2013; Zhang et al., 2011). Therefore, the rapid spread and

3

replacement of GI viruses might depress the protective efficacy induced by current JE

4

vaccines, and an improved JE vaccine that would confer broad protection against all

5

circulating genotypes, especially GI viruses, is needed.

6

Flavivirus E glycoprotein is the principal antigen in eliciting neutralizing

7

antibodies and plays a critical role in inducing protective immunity against virus

8

infection. The E protein contains three discernible domains (DI, DII and DIII) (Luca

9

et al., 2012). Numerous neutralizing epitopes have been identified at the tip of DII

10

(Oliphant et al., 2006), in the hinge region between DI and DII (de Alwis et al., 2012;

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Teoh et al., 2012), and on the DIII (Austin et al., 2012; Gromowski & Barrett, 2007;

12

Oliphant et al., 2005). Previous investigation on DENV has revealed genotype- and

13

strain-dependent epitopes that impacts neutralization and protection (Shrestha et al.,

14

2010; Sukupolvi-Petty et al., 2013).

15

In the present study, by combining bioinformatic analysis and reverse genetic

16

technology, we identified two JEV genotype-specific neutralization determinants

17

within E protein (E222 and E327). And the corresponding mutations (A222S and

18

S327T) were rationally engineered into the LAV strain SA14-14-2 to generate an

19

improved version of JE LAV with enhanced neutralization capability against GI

20

circulating strains.

21 22 23

RESULTS

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E222 and E327 were distinct in JEV GI and GIII strains. A total of 40

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envelope gene sequences of JEV strains were retrieved from GenBank, the detailed

26

information about the origin, collection date and host of the JEV strains used in this

27

study was shown in Table S1. Phylogenetic analysis and multiple sequence alignment

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of E protein identified two genotype-specific amino acid residues (Fig. 1a and b). The

29

amino acid 222 and 327 of E protein (E222 and E327) are alanine (A) and serine (S)

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in all GIII strains including LAV SA14-14-2 respectively, while all GI strains contain 5

1

serine (S) and threonine (T) at the same positions, respectively. Besides, E222 is

2

serine (S) in GII viruses, or alanine (A) in GIV and GV viruses; E327 is threonine (T),

3

leucine (L) or glutamine (Q) in GII, GIV and GV viruses, respectively. Then, all

4

available sequences of complete E gene of JEV were retrieved from GenBank, and

5

E222 and E327 were confirmed as GI- and GIII- specific in all 570 E protein

6

sequences (Fig. S1).

7

To figure out the three dimensional location of E222 and E327, the two residues

8

were mapped to the crystal structure of JEV E protein (Fig. 1c) and the reconstructed

9

virion (Fig. 1d). It was found that E222 and E327 locate in DII and DIII, respectively.

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E327 was clustered with other epitopes recognized by known neutralizing antibodies

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(Fig. 1c). The reconstructed virion model revealed that E327 is largely exposed on the

12

surface of mature virion, while E222 was buried inside (Fig. 1d), suggesting that these

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two amino acids may function in different mechanisms.

14

A222S and S327T mutations decreased LAV-induced neutralization. To

15

clarify the potential role of E222 and E327 in neutralization, single and double

16

mutations of these two residues were introduced into a full-length infectious clone of

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GIII JEV (pAJE70). After standard in vitro transcription and transfection procedure,

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all three mutant viruses (A222S, S327T, and A222S/S327T) were recovered in

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BHK-21 cells. RT-PCR and sequence analysis confirmed that the corresponding

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mutations were successfully engineered into JEV genome. Immunofluoscence

21

staining using anti-JEV mouse sera showed that A222S, S327T or A222S/S327T

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generated similar levels of viral protein expression with the WT virus (Fig. 2a). There

23

is no significant difference in plaque morphology among the four viruses (Fig. 2b).

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Growth curves in BHK-21 cells showed that WT and the mutant viruses A222S,

25

S327T, and A222S/S327T replicated efficiently, and peaked at 48 h post-infection

26

with titers of 106.94 PFU/ml, 106.40 PFU/ml, 106.79 PFU/ml and 106.69 PFU/ml,

27

respectively (Fig. 2c). These results demonstrated that A222S and S327T mutations in

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E protein did not significantly impact viral replication.

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Then, to clarify whether these mutations affect JEV neutralization, a panel of

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human sera from volunteers immunized with LAV SA14-14-2 was used to assess the 6

1

neutralizing antibody titers against WT and each mutant virus. As shown in Fig. 3, all

2

human sera could efficiently neutralize the three mutant viruses together with the WT

3

virus (Fig. 3). However, the PRNT50 titers against each mutant virus significantly

4

decreased in comparison with that against the WT virus for selected sera (#1, #2, and

5

#5). These results showed that single mutation in either E222 or E327 would affect

6

the neutralization capability against GI and GIII JEV.

7

LAV-A222S and LAV-S327T induced higher neutralizing antibody titers

8

against GI strains. Further, to clarify whether these genotype-specific mutations

9

would enhance the neutralization capability of LAV SA14-14-2 against GI viruses,

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A222S and S327T mutation was individually introduced into the infectious clone of

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LAV SA14-14-2 by standard reverse genetic approach. As expected, the resulting two

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mutant viruses, named LAV-A222S and LAV-S327T, respectively, were successfully

13

rescued with similar viral protein expression efficiency (Fig. 4a) and plaque

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morphology (Fig. 4b) in BHK-21 cells in comparison with LAV. Growth kinetics in

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BHK-21 and C6/36 cells demonstrated that LAV-A222S and LAV-S327T replicated

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similarly with LAV SA14-14-2 in mammalian cells but exhibited a slightly decreased

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multiplication capacity in mosquito C6/36 cells (Fig. 4c).

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The immunogenicity of LAV-A222S and LAV-S327T were further tested in mice

19

in comparison with LAV SA14-14-2. Groups of 3-week-old mice were immunized

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with equal dose of LAV, LAV-A222S or LAV-S327T, respectively, and neutralizing

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antibody response against representative GI circulating strains SX06 and SH53 were

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determined, respectively. The results showed that both LAV-A222S and LAV-S327T

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could elicit higher titers of neutralization antibodies against GI strains SX06 and

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SH53 than that by SA14-14-2 (Fig. 5a and b). These results further demonstrated the

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introduction of A222S or S327T mutation into the LAV SA14-14-2 enhanced the

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neutralization capacity against GI strains.

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DISCUSSION

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In our study, phylogenetic analysis and sequence alignment of all available JEV E

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protein sequences revealed E222 and E327 are conserved within, meanwhile distinct 7

1

between GI and GIII viruses. The introduction of mutations into the two residues

2

significantly changed the neutralization capability of human sera, demonstrating the

3

critical role of E222 and E327 in JEV genotype-specific neutralization. To our

4

knowledge, E222 and E327 are first reported as the only known genotype-specific

5

neutralization determinants of JEV. Structural modeling revealed that E327 was in the

6

lateral ridge in DIII and largely exposed on the mature virion (Fig. 1c and d),

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clustered with the residues (G302, E306, S331, D332, G333, I337, F360 and R387)

8

recognized by JEV-specific neutralizing MAbs (Cecilia & Gould, 1991; Goncalvez et

9

al., 2008; Lin & Wu, 2003; Wu et al., 2003; Wu et al., 1997) , indicating that E327

10

probably directly influences neutralization by interacting with other identified

11

neutralizing epitopes nearby. Structure modeling indicated that E222 is not exposed

12

on the surface of mature JEV virion (Fig. 1d), whether any conformational interaction

13

with the identified neutralizing epitopes remains unknown. In addition, structural

14

analysis revealed the diversification of flavivirus surface structure in virus life-cycle,

15

and the hidden epitopes may expose on the surface through the conformational

16

changes in viral glycoprotein arrangement (Lok et al., 2008; Zhang et al., 2004).a

17

These genotype-dependent epitopes are JEV specific, and completely different

18

from that of DENV (Bernardo et al., 2009; Brien et al., 2010; Kochel et al., 2002;

19

Shrestha et al., 2010; Wong et al., 2007). Previous studies using monoclonal

20

antibodies indicated that the potent neutralizing capacity was impacted by the

21

genotype-dependent conformational change/exposure of the CC’ loop epitope in DIII

22

(Austin et al., 2012). The natural occurring genotype- or strain- dependent amino acid

23

variations in DIII have been demonstrated to affect the binding and neutralization of

24

type-specific MAbs of DENV (Pitcher et al., 2012; Sukupolvi-Petty et al., 2013;

25

Wahala et al., 2010).

26

The LAV SA14-14-2 possesses advantages of satisfactory immunogenicity, less

27

dosage of vaccination and low cost of production compared to inactivated vaccine. It

28

now takes the largest JE vaccine market worldwide, and further improvement in

29

vaccine efficacy would be meaningful. Based on the infectious cDNA clone of LAV

30

SA14-14-2, the newly identified genotype-specific neutralization determinants were 8

1

rationally engineered. The mutant viruses LAV-A222S and LAV-S327T showed

2

similar plaque morphology and replicated efficiently in Vero cells with peak titers of

3

105.9 PFU/ml and 106.5 PFU/ml (data not shown), indicating the potential capability of

4

large-scale production and industrialization at low cost. Both mutants exhibited

5

potential genetic stability after serial passages in Vero cells (data not shown). For

6

immunogenicity test, immunization with LAV-A222S and LAV-S327T were capable

7

of eliciting protective neutralizing antibody against the circulating GI viruses SX06

8

and SH53, with increased antibody titers compared to those induced by native LAV

9

SA14-14-2. These properties support LAV-A222S and LAV-S327T as an improved

10

version of JEV LAV.

11

Until now, the mechanisms of circulating JEV genotype replacement are not fully

12

understood. E protein is the phylogenetic basis for JEV genotype division and the

13

major component on virion surface as well as the critical factor of virulence and

14

immunity, therefore it is considered as one of the most important subject investigated.

15

Phyletic and adaptive evolutionary analyses predicted the positively selected sites

16

within GI and GIII alignment as potential genetic determinants affecting the genotype

17

distribution, besides, it has been suggested that increased viral multiplication in avian

18

and mosquito as well as the more efficient replication and transmission cycle may be

19

responsible for the genotype replacement (Han et al., 2014; Schuh et al., 2014). In

20

this study, the recovered JE viruses LAV-A222S and LAV-S327T with GI mutations

21

showed a slightly decrease in replication efficiency in C6/36 cells, and the possible

22

role of host adaptions remains to be determined. Moreover, naturally occurring

23

sequence variation within or between genotypes may influence the antibody binding

24

and neutralization capacity (Austin et al., 2012; Sukupolvi-Petty et al., 2013; Wahala

25

et al., 2010). More data of antigen variation surveillance and evolution analysis are

26

required for the explanation of the genotype shift.

27

Our results have important implications for the evaluation and design of next

28

generation JE vaccine candidate. Currently, the JE chimeric live attenuated vaccine

29

JE-CV has been used in Asian countries as well as SA14-14-2 (Bonaparte et al., 2014;

30

Feroldi et al., 2014), and several flavivirus chimeric live attenuated vaccine carrying 9

1

JEV E protein is under development (Gromowski et al., 2014; Sjatha et al., 2014).

2

These two amino acid mutations A222S and S327T should be taken into consideration

3

for the rational design and improvement of a new JEV vaccine with potential

4

protection capability against all circulating JEV strains.

5 6

MATERIALS AND METHODS.

7

Cells and viruses. BHK-21 and Vero cells were cultured in Dulbecco’s modified

8

Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum

9

(FBS) in growth medium in 5% CO2 environment at 37℃. C6/36 cells were grown in

10

RPMI 1640 (Invitrogen) medium supplemented with 10% FBS at 37℃ in 5% CO2.

11

LAV strain SA14-14-2 (GIII) was from Chengdu Institute of Biological Products, and

12

its parental strain SA14 was from National Institute for Food and Drug Safety. GI JEV

13

strains SX06 and SH53 were isolated in Shanxi, in 2006, and Shanghai, in 2001,

14

respectively (Ye et al., 2014). All JEV strains were propagated in BHK-21 cells

15

cultured in DMEM with 2% FBS. Virus stocks were stored in aliquots at −80℃ until

16

use. Virus titers were determined by standard plaque forming assay in BHK-21 cells

17

as described before (Li et al., 2013a).

18

Phylogenetic analysis and sequence alignment. A total of 40 E protein gene

19

sequences of JEV strains (Table S1), which were isolated from a variety of geographic

20

regions in Asia, were collected from GenBank.

21

Multiple sequence alignment (MSA) and phylogenetic analysis were performed

22

by CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and MEGA

23

version 5.0 software (http://www.megasofteware.net). The phylogenetic tree was

24

constructed by the neighbor-joining method and the maximum composite likelihood

25

model.

26

Structure modeling. The crystal structure of JEV SA14-14-2 E protein (PDB

27

accession code, 3P54) was used to exhibit the modeled structure of the E protein.

28

Considering the similarity among mosquito borne flaviviruses, the E protein monomer

29

was superimposed onto the DENV-2 virion model (PDB accession code, 1THD)

30

based on cryo-EM reconstruction to generate a rational model for JEV virion. All the 10

1

identified JEV neutralizing epitopes were mapped onto JEV E protein and the

2

modeled virion (Luca et al., 2012). The structure demonstration and graphic rendering

3

were performed by using PyMOL.

4

Construction and characterization of recombinant JEV. All plasmids were

5

constructed using standard molecular clone protocols and confirmed by DNA

6

sequencing. Single mutation (A222S or S327T) or both mutations (A222S and S327T)

7

in E protein were introduced into the infectious full-length JEV (GIII) cDNA clone

8

pAJE70 previously described (Ye et al., 2012) by site-directed mutagenesis based on

9

overlapping PCR. The constructed plasmids were named pAJE70-A222S,

10

pAJE70-S327T and pAJE70-A222S/S327T, respectively. The mutations mentioned

11

above were introduced into the infectious full-length cDNA clone of LAV SA14-14-2

12

(named LAV) in the same fashion.

13

Xho I-linearized plasmids were purified and used as DNA templates for in vitro

14

transcription as previously described (Li et al., 2013a) in the presence of m7GpppA

15

cap analog with RiboMaxTM Large Scale RNA Production system (Promega). RNA

16

transcripts were transfected into BHK-21 cells with Lipofectamine 2000 (Invitrogen).

17

Four days after transfection, culture supernatants were harvested and seeded into

18

BHK-21 monolayer to prepare virus stocks. Recovered viruses derived from pAJE70,

19

pAJE70-A222S, pAJE70-S327T and pAJE70-A222S/S327T were named WT, A222S,

20

S327T and A222S/S327T. Mutant viruses based on LAV SA14-14-2 were named

21

LAV-A222S and LAV-S327T, respectively.

22

Indirect immunofluorescence assay were performed in BHK-21 cells using

23

anti-JEV mouse sera as previously described (Li et al., 2013b). Growth curves of all

24

recombinant viruses were generated by infecting confluent BHK-21 and C6/36 cells

25

in a 24-well plate. Cell supernatants were collected per 24 h post-infection and virus

26

titers were determined by standard plaque assay in BHK-21 cells.

27

Neutralization assay. A panel of human serum samples were collected from

28

volunteers vaccinated with LAV and subjected to 50% plaque reduction neutralization

29

test (PRNT50) (Ye et al., 2014). Briefly, 2-fold serial dilutions of serum sample were

30

inactivated at 56℃ for 30 min, and then mixed with equivalent JEV suspension and 11

1

incubated at 37℃ for 90 minutes. The PRNT50 titers were calculated by the method of

2

Karber (Traggiai et al., 2004). PRNT50 >1:10 was considered protective (Hombach et

3

al., 2005).

4

Animal immunization. To evaluate the immunogenicity of mutant vaccines,

5

groups of 3-week-old female BALB/c mice were injected by s.c. route with equal

6

dose (105 PFU/mouse) of LAV-A222S or LAV-S327T, and LAV group was set as

7

control. A booster dose was given 30 days after primary immunization, and mice sera

8

were collected 30 days post the second immunization. Sera samples were inactivated

9

and stored at -20℃ and subjected to neutralization assay by PRNT50.

10 11

Statistic analysis. Significant difference of neutralization titers was analyzed by one-way ANOVA using Graphpad Prism 5.0.

12 13 14 15

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No.31300151 and No.31470265).

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Figure legends

3

Fig. 1. E222 and E327 were distinct in JEV GI and GIII strains. (a)

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Neighbor-Joining Tree was constructed by Tamura-Nei model with gamma-

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distribution of among-site with 40 envelope gene sequences of JEV isolates. (b)

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Sequence alignment of representative GI and GIII strains. The genotype specific

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residues 222 and 327 were highlighted in multicolor. (c) Structural model of JEV

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monomer E protein. Domain I, II and III were colored in palecyan, yellow and blue,

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respectively. All the identified neutralizing epitopes were colored in green, and the

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genotype specific neutralization epitope E222 and E327 in envelope protein were

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colored in red and hotpink, respectively. (d) The pseudo-virion model of JEV based

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on the reconstruction of DENV-2 cryo-EM density map.

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Fig. 2. Recovery and characterization of A222S and S327T mutants. (a) Indirect

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immunofluorescence assay of WT, A222S, S327T and A222S/S327T viruses infected

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BHK-21 cells. The expression of E protein was detected using anti-JEV mouse sera at

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24 h and 48 h post-infection. (b) Plaque morphology of WT, A222S, S327T and

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A222S/S327T in BHK-21 cells. (c) Growth curves of WT, A222S, S327T and

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A222S/S327T in BHK-21 cells. BHK-21 cells were infected with viruses at an MOI

19

of 0.01. Culture supernatants were harvested at 24, 48 and 72 h post-infection. Virus

20

titers were determined using standard plaque assays in BHK-21 cells and shown as

21

mean±SD from two independent experiments.

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Fig. 3. Neutralizing antibody titers with LAV SA14-14-2 immunized human sera.

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PRNT50 titers of human sera against WT, A222S, S327T and A222S/S327T were

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shown in histogram as mean±SD from two independent experiments. The statistical

25

significance was analyzed by one-way ANOVA and indicated by asterisks (* P

Genotype-specific neutralization determinants in envelope protein: implications for the improvement of Japanese encephalitis vaccine.

Japanese encephalitis remains the leading cause of viral encephalitis in children in Asia and is expanding its geographical range to larger areas in A...
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