JGV Papers in Press. Published August 28, 2014 as doi:10.1099/vir.0.067736-0

Infection of influenza virus NA vaccinated mice with homologous influenza virus leads to strong protection against heterologous influenza viruses

Biao He1, Haiyan Chang1, Zhihua Liu1, Chaoyang Huang1, Xueying Liu2, Dan Zheng2, Fang Fang1, Bing Sun3 and Ze Chen1,2 * 1

College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan,

China 2

Shanghai Institute of Biological Products, Shanghai 200052, China

3

Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese

Academy of Sciences, Shanghai 200025, China Footnotes: *

Correspondence should be addressed to Ze Chen

E-mail: [email protected]; [email protected] Phone/Fax: (00) 86-(0) 21-62826658 Address: Shanghai Institute of Biological Products, Shanghai 200052, China

Running title: NA vaccine and influenza infection lead to protection The Contents Category for the paper: Animal Viruses – Negative-strand RNA The word count of the summary: 217 The word count of the main text: 5409 The number of tables and figures: 8

1

1

Summary

2

Vaccination is the best measure to prevent influenza pandemics. Here we studied the

3

protective

4

A/reassortant/NYMC

5

A/Chicken/Jiangsu/7/2002 (H9N2) and A/Guizhou/54/89xA/PR/8/34 (A/Guizhou-X)

6

(H3N2), in mice first vaccinated with a DNA vaccine of hemagglutinin (HA) or

7

neuraminidase (NA) of A/PR/8/34 (PR8) and then infected with the homologous

8

virus. We showed that PR8 HA or NA vaccination both protected mice against a

9

lethal dose of the homologous virus; PR8 HA or NA vaccination and then PR8

10

infection in mice respectively offered poor or excellent protection against a second,

11

heterologous influenza virus challenge. In addition, before the second heterologous

12

influenza infection, the highest antibody level against the NP, M1 and M2 was found

13

in PR8 NA DNA vaccinated and PR8 infected group. The level of induced cellular

14

immunity against NP and M1 showed a trend consistent with that seen in antibody

15

levels. However, PR8HA+NA and then PR8 infection resulted in limited protection

16

against heterologous influenza virus challenges. Results of the present study

17

demonstrated that infection of the homologous influenza virus in mice already

18

immunized with a NA vaccine could provide excellent protection against subsequent

19

infection of a heterologous influenza virus. These findings suggest that NA, a major

20

antigen of influenza virus, could be an important candidate antigen for universal

21

influenza vaccines.

22

Keywords: influenza; hemagglutinin; neuraminidase; cross-protection

effect

against X-179A

heterologous (pH1N1),

23 2

influenza

viruses,

A/chicken/Henan/12/2004

including (H5N1),

24

Introduction

25

In 2009, a new influenza A (H1N1) virus epidemic broke out in humans worldwide.

26

This virus was easy to spread among humans and resulted in infections in a large

27

number of people. Fortunately, the mortality was not high (Garten et al., 2009;

28

Neumann et al., 2009). The influenza A (H5N1) virus also has brought huge threat to

29

humans; the virus has high mortality in humans but has difficulty to transmit among

30

humans and has not caused a pandemic (Sambhara and Poland, 2010).

31

In addition, in March of 2013, the first influenza A (H7N9) virus human infection

32

occurred in China (Gao et al., 2013). So far a total of 355 persons have been

33

confirmed H7N9 infection, of which 112 died (World Health Oganization,2014).

34

There has not been any solid evidence to demonstrate that H7N9 is capable of

35

human-to-human transmission. However, given the high mutation rates of influenza

36

viruses, it is possible that an influenza virus with high mortality and high

37

transmissibility will reemerge and bring a huge disaster to humans.

38

Currently the most effective method for preventing influenza virus infection is

39

influenza vaccination. Influenza virus surface proteins hemagglutinin (HA) and

40

neuraminidase (HA) are the most important antigens for eliciting immune responses

41

(Johansson et al., 1989). We have reported that in mouse models, DNA vaccines

42

based on influenza virus HA and NA proteins could provide well protection against

43

the homologous influenza virus, and the protective effects were better than other

44

influenza viral proteins (Chen et al., 2009; Chen et al., 2000; Chen et al., 1998).

45

However, HA and NA-based influenza virus vaccines have limited protection against

3

46

heterologous viruses. The influenza virus NP, M1 and M2 proteins are highly

47

conserved, and vaccines based on these proteins showed some protection against

48

heterologous influenza viruses in mouse models (Epstein et al., 2005; Guo et al.,

49

2010; Neirynck et al., 1999; Sui et al., 2010a; Sui et al., 2010b). Existing influenza

50

vaccines mainly rely on HA and NA of influenza viruses. The World Health

51

Organization must regularly change vaccine composition in response to immune

52

escape of HA and NA proteins (Carrat and Flahault, 2007), which not only increases

53

the burden of vaccine production, but also places people in a passive position in the

54

face of new influenza viruses.

55

Previously, it has been reported that influenza virus infection could result in

56

cross-protection. For example, H3N2 infection of mice could induce protective

57

immunity against H5N1 (Kreijtz et al., 2009). In another study, infection of mice with

58

H1 and H3 influenza viruses could protect them against a challenge by the

59

heterologous 2009 H1N1 A/CA/04/09 influenza virus, and it has been demonstrated

60

that the protection is mediated by T-cell immunity (Guo et al., 2011). In addition,

61

cross-protection mediated by T cell immunity against influenza viruses has also been

62

confirmed in pigs, chickens, ducks and ferrets (Carter et al., 2013; Fereidouni et al.,

63

2009; Heinen et al., 2001; Seo and Webster, 2001).

64

In the present study, we demonstrated in mouse model that vaccination with a DNA

65

vaccine of influenza virus NA gene could not only protect mice against infection by

66

the homologous influenza virus, but also protect well against infection by a second,

67

heterologous influenza virus. However, mice vaccinated with a DNA vaccine of

4

68

influenza virus HA gene offered limited protection against infection of a second,

69

heterologous influenza virus. These results suggest that NA, a major antigen of

70

influenza virus, could be used as an important candidate antigen for universal

71

influenza vaccines.

72 73

Results

74

Protection of DNA vaccines based on PR8 HA, NA, and HA+NA against a lethal

75

dose of PR8 infections

76

Mice were divided into eight groups. The group names, vaccination contents and

77

schedule, and challenge schedule are shown in Table 1. On day 3 and 5 after PR8

78

infections, mice lung lavage fluids were collected for virus titer detection. The

79

survival rates for the HAP, NAP and HNP groups was all 100 % (Table 2), and no

80

symptoms of influenza virus infection were observed. All of the C1 group mice died

81

within 10 days post-infection. For the lung virus load at day 3 post infection, no

82

influenza virus was detected in MDCK cells using lung lavage from the HAP and

83

HNP group, while lung virus titer for the NAP group and C1 group mice were 1.4 ±

84

0.1 log10TCID50/ml and 4.35 ± 0.35 log10TCID50/ml, respectively, but the titer in NAP

85

group was significantly lower (P