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Vaccine 32 (2014) 5375–5378

Contents lists available at ScienceDirect

Vaccine

j our na l ho me page: www.elsev ier .com/ locate /vacc ine

rief report

omplete protection against lethal challenge of novel H7N9 virusith heterologous inactivated H7 vaccine in mice

ay Hoon Tanga, Jie Hui Lima, Li Fang Kuaha, Yuk Fai Laua,b,∗

Host-pathogen Interactions Laboratory, DMERI, DSO National Laboratories, 27 Medical Drive, 117510, Republic of SingaporeProgram in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Road, 169857, Republic of Singapore

r t i c l e i n f o

rticle history:eceived 22 October 2013eceived in revised form 29 April 2014

a b s t r a c t

A prototype H7 influenza vaccine constructed based on the H7N7 outbreak in 2003 was tested for theprotective efficacy against the novel H7N9 virus in a lethal murine challenge model. Serum samplesfrom vaccinated mice showed significant neutralizing activity against the H7N9 virus and the mice were

ccepted 31 July 2014vailable online 15 August 2014

eywords:nfluenza7N9

completely protected with no significant weight loss. The results have direct implications on how toovercome potential vaccine shortage and identify donors for immune sera for passive immunization.

© 2014 Elsevier Ltd. All rights reserved.

accine

. Introduction

As of August 2013, there have been 135 cases of H7N9uman infection with 44 fatalities (WHO Website http://www.ho.int/influenza/human animal interface/influenza h7n9/Data

Reports/en/). Outbreaks of H7 viruses have occurred sporadicallyn humans [1]. There was an outbreak of H7N7 virus in Netherlands

ith a fatal case in 2003 [2]. With its potential of crossing thepecies-barrier, various H7 vaccines have been developed andested in various animal models [3–5]. Two live attenuatednfluenza vaccines (LAIV) that contain the hemagglutinin (HA)nd neuraminidase (NA) of A/chicken/BC/CN-6/04 (H7N3) and/Netherlands/219/03 (NL/03, H7N7) were evaluated in micend ferrets and two doses were required for optimal protectiongainst viruses from the North American and Eurasian lineages3,5]. When tested in humans, the H7N3-LAIV was immunogenicith 48% volunteers showing an increase in micro-neutralization

MN) titers against the vaccine virus [6]. As for inactivatedaccine, when co-administered with immune stimulatory com-lexes (ISCOMs), the adjuvanted subunit vaccine that contains

he HA of A/Mallard/Netherlands/12/00 (H7N3) reduced viraliter by 1000-fold when the vaccinated mice were challengedith NL/03 wild-type (wt) virus [7]. However, when tested as

∗ Corresponding author at: Host-pathogen Interactions Laboratory, DMERI, DSOational Laboratories, 27 Medical Drive, 117510, Republic of Singapore.el.: +65 64857077; fax: +65 64857226.

E-mail address: lyukfai@dso.org.sg (Y.F. Lau).

ttp://dx.doi.org/10.1016/j.vaccine.2014.07.087264-410X/© 2014 Elsevier Ltd. All rights reserved.

an un-adjuvanted vaccine in humans, the subunit vaccine waspoorly immunogenic [8]. Since the hemagglutinin (HA) of theH7N9 virus shares significant homology with other H7 HAs (95%homology in amino acid sequence between KC885956 (H7N9) andAY338459 (H7N7)). It is of interest to determine if heterologous H7vaccine-induced immunity could provide cross-protection againstthe H7N9 virus. In the event of a lack of homologous vaccine,the results provide important information on alternative vaccineseed source, as well as donor identification for serum therapy foremergency situations.

2. Materials and methods

2.1. Virus

RG104 is a (7 + 1) reassortant virus with a A/PR/8/34 (PR8)background, containing the HA gene of NL/03 wt virus (with theremoval of polybasic amino acid motif), generated using tech-nique previously described [9]. The virus stocks were propagatedin embryonated hen’s eggs. The wt A/Anhui/1/2013 (AH/13) H7N9virus is provided by Drs. Eri Nobusawa and Masato Tashiro atNational Institute of Infectious Diseases, Japan and amplified usingMDCK cells. The virus stocks were aliquoted and stored at −80 ◦C.

2.2. Vaccine

Inactivated whole virion RG104 vaccine was prepared by inacti-vation of MDCK-derived virus with 4% paraformaldehyde for 3 days,followed by overnight dialysis in PBS at 4 ◦C. Inactivated whole

5376 L.H. Tang et al. / Vaccine 32 (2014) 5375–5378

Fig. 1. Protection against H7N9 virus by immune responses induced by a heterologous H7 virus. Groups of 5 BALB/c mice were primed with indicated viruses i.p. and receiveda boost 3 weeks later (2 × 105 TCID50 per injection, equivalent to 25 and 3 HA units for RG104 and PR8 respectively). Ten weeks after the boost, the mice were challengedw eight loss (A) and survival (B). Mice that lost >25% of initial weight were euthanized. Ana statistically significant (p < 0.05). Seroconversion of PR8-primed mice against PR8 virusw

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Fig. 2. Cross-neutralization of viruses by antibodies induced by inactivated RG104vaccine. Groups of 5 BALB/c mice were primed with 80 HA unit of formalin-inactivated RG104 vaccine with or without PIKA adjuvant (100 �g) and boosted onday 25. Serum samples were collected on day 24 and 89 and tested for neutralizingantibody titers against the two viruses. Each dot represents the titer of each mouse

ith 50 LD50 of AH/13 virus. Mice were monitored for 2 weeks after challenge, for wsterisk indicates that the difference between the RG104 group and PBS group wasas confirmed by ELISA and MN assay.

irion H7N9 vaccine was similarly prepared in the BSL3 and after 2erial passages in MDCK cells to confirm inactivation, the vaccineas downgraded to BSL2 for dialysis. Vaccine protocols are simi-

ar to those previously described [10]. PIKA adjuvant is providedy Yisheng Biopharma Singapore. The animal study protocols usedere approved by the Institutional Animal Care and Use Commit-

ee of DSO National Laboratories and were conducted at the ABSL2nd ABSL3 laboratories at DMERI.

.3. Micro-neutralization assay

The assay was performed as previously described [10].

.4. Statistical analysis

The significance of difference between any two differentroups was assessed by a Mann–Whitney test using Prism, ver-ion 5 (GraphPad Software, CA). Kaplan–Meier log rank test waserformed on survival data. p values of <0.05 are considered signif-

cantly different.

. Results and discussion

RG104 is a reassortant virus that contains the HA gene ofL/03 wt virus with a PR8 background. To evaluate the antigenic

elationship between the RG104 and H7N9 viruses, groups of 5ALB/c mice were inoculated by intraperitoneal (i.p.) injection with

× 105 TCID50 of RG104 virus. The mice were boosted 3 weeksater. Ten weeks after the boost, the mice were challenged with.6 × 105 TCID50 of AH/13 (H7N9) virus which is ∼50 LD50 (50%

ethal dose) [11].As shown in Fig. 1A and B, compared with the mock controls,

he RG104-primed mice showed insignificant weight loss after theethal challenge (p < 0.05) and returned to initial weight by day

post-infection (p.i.) with 100% survival. PR8-primed mice weresed to control for any cellular immunity that could protect theice through recognizing the internal conserved epitopes. Theseice showed similar weight loss as mice that received PBS and

nly 1 mouse survived the lethal challenge, hence showing signif-cantly lower survival rate than RG-104 primed mice (p = 0.0143).he results suggest that the humoral immunity induced by RG104

irus played a significant role in mediating protection against the7N9 virus.

To further strengthen the hypothesis that the humoralesponses induced by RG104 could protect against the wt H7N9

and the geometric mean titer of the groups are represented by the lines. No signif-icant difference was observed between the adjuvanted group and non-adjuvantedgroup at any of the time-points examined.

virus, groups of BALB/c mice were vaccinated with formalin-inactivated RG104 with an adjuvant (PIKA) subcutaneously (s.c.).The mice were boosted 25 days later. Serum samples collected atvarious time-points were tested against the H7N9/RG104 viruses ina standard MN assay. As shown in Fig. 2, after 1 dose of inactivatedRG104, the mice developed detectable level of neutralizing anti-bodies against both the RG104 and H7N9 viruses. Boosting withan additional dose further enhanced the responses. Although theRG-104 induced serum samples gave higher antibody titers againstthe homologous virus (RG-104) than to the heterologous H7N9virus, the difference in the MN titers did not reach statistical signif-icance for the 4 data pairs (p = 0.2492, 0.0593, 0.1706 and 0.0545).Although the effect of including an adjuvant was more apparentduring the primary response, the difference did not reach statistical

significance (p = 0.0593 for RG104 and 0.1161 for H7N9). For post-boost responses, including PIKA did not lead to a significant increasein antibody titers (p = 0.4529 for RG104 and 0.8991 for H7N9). Theuse of inactivated whole virion vaccine at a relatively high dose of

L.H. Tang et al. / Vaccine 32

Table 1Cross-neutralizing antibody responses against the two H7 viruses. The post-boostserum samples from Fig. 2 (n = 5 per group) were tested for their cross-neutralizingability against RG104 and AH/13 viruses in a standard micro-neutralizing assay.The geometric mean and S.D. of the groups are presented. The difference of theMN titers of the indicated immunogen against the two viruses was analyzed byMann–Whitney test and the p values are listed. This is representative of two inde-pendent experiments.

Virus

RG104 H7N9 p value

Immunogen

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RG104 10.3 ± 1.1 8.6 ± 0.8 0.0545H7N9 6.7 ± 2.2 9.6 ± 0.3 0.0117

0 HA units (compared to 3 HA units of PR8 in Fig. 1) could be theeason for the good immunogenicity of this un-adjuvanted vaccine.

A cross micro-neutralizing assay was performed to determinentigenic relationship between the two H7 viruses. As shown inable 1, the RG104 vaccine-induced serum samples showed signif-cant cross-reactivity against the novel H7N9 virus and the average

as not statistically different from the homologous pair (p > 0.05).he experiment was repeated with an additional 10 mice andheir MN titers against RG104 and AH/13 were also not statisti-ally different (8.81 ± 0.92 and 7.94 ± 1.69 respectively, p = 0.4027).dditionally, mice vaccinated with the inactivated H7N9 vaccineith 2 additional boosts also showed good neutralizing titers

gainst the wt H7N9 virus, suggesting good antigenicity of the virusA. However, the serum samples showed significantly lower neu-

ralizing activity against the RG104 virus (p = 0.0117).To determine the biological relevance of the observed cross-

eutralizing activity of the serum samples, the RG104-vaccinatedice were challenged with the H7N9 virus 10 weeks after the final

oost. As shown in Fig. 3, all mice that received inactivated RG104,ith or without PIKA, survived challenge with a significantly higher

urvival rate than the PBS group (p = 0.0023). Mice that received

he adjuvanted vaccine showed no significant weight loss afterhe lethal challenge, whereas mice that received the unadjuvantedaccine showed moderate weight loss, which reached statisticalignificance (p < 0.05) on days 4–6, 8 and 14. Finally, mice that

ig. 3. Protection against lethal challenge of H7N9 virus by inactivated RG104 vaccine. Tfter the boost. As a positive control, 5 mice were primed with 160 HA units of formalinhese mice were challenged 4 weeks after the final boost. All mice listed in this figure

urvival (A) and weight loss (B). Mice that lost >25% of initial weight were euthanized. AnG104 vaccine and unadjuvanted vaccine reached statistical significance (p < 0.05).

(2014) 5375–5378 5377

received the H7N9 vaccine remained healthy throughout the exper-imental period with no significant weight loss. Between the RG104and H7N9-vaccinated mice, weights were comparable except onday 8 (p = 0.0317). In conclusion, the cross-neutralizing antibodyresponses have biological relevance in that the level of protectionachieved by the adjuvanted RG104 vaccine was comparable to micethat received the homologous inactivated H7N9 vaccine.

Based on the MN results, the RG104-induced humoral immu-nity shows good level of cross-reactivity against AH/13 (Table 1),suggesting there is antigenic similarity between the two viruses.In a recent study by Xu et al. [12], NL/03-immunized ferret seracross-reacted with AH/13 in both hemagglutination inhibitionand micro-neutralization assays. The cross-reactivity was ∼3-foldlower than the homologous pair. Due to the lack of a BSL3 facil-ity, the authors did not perform a challenge study. In this report,we demonstrate that the NL/03-primed mice were protected fromweight loss and mortality against the novel H7N9 virus in a lethalchallenge model. The high titer of neutralizing antibodies in theserum observed in this study (Table 1) is likely to be the mediatorfor the protection as Min et al. previously demonstrated that pas-sive transfer of serum antibodies elicited by NL/03 HA-containingvaccine protect mice from challenge of heterologous H7 viruses [3].

While vaccine manufacturers have already looked into gener-ating vaccine seeds for this novel H7N9 virus, there is a significantbody of work that has been performed in evaluating the antigenic-ity and safety profiles of various H7 vaccines in different animalmodels [3–5], as well as clinical trials in humans [6,8]. Some ofthese vaccines might have been manufactured and stockpiled as apart of the preparation plan for a pandemic. In the event of short-age of homologous vaccine, the information described in this studycan provide supportive evidence for the use of heterologous vac-cines to alleviate a sudden surge in vaccine demands, especiallyfor the first-line responders who need to be vaccinated promptly.Additionally, serum samples that have been stored from various H7vaccine trials should be re-examined for their neutralizing activity

against the H7N9 virus. Together with the patients who recoveredfrom the H7N9 infections, these vaccine trial volunteers could be avaluable source of immune serum for passive immunization. Stock-piling immune serum from these individuals can be life-saving as

he mice described in Fig. 2 were challenged with 50 LD50 of AH/13 virus 10 weeks-inactivated H7N9 vaccine with 100 �g of PIKA s.c. and boosted on day 14 and 28.were challenged at the same time and monitored for 2 weeks after challenge, for

asterisk indicates that the difference between the groups that received adjuvanted

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[15] Khurana S, Verma N, Yewdell JW, Hilbert AK, Castellino F, Lattanzi M, et al.MF59 adjuvant enhances diversity and affinity of antibody-mediated immune

378 L.H. Tang et al. / Vac

uch treatment has been shown to give promising results in treatingevere viral infections in humans, including H5N1 [13] and sH1N114]. Finally, adjuvants have been shown to improve the diver-ity, affinity and isotype distribution of vaccine-induced humoralesponse [10,15]. These factors could explain our observation thatven though the humoral responses induced by adjuvanted or non-djuvanted vaccines were comparable (Fig. 2), mice that receiveddjuvanted vaccine were better protected (Fig. 3) during challenge.

recent clinical trial reported that an H7N9 virus-like-particleaccine is poorly immunogenic in humans, with only 5.7% serocon-ersion rate after 2 doses of 15 �g of HA. With the incorporation ofhe ISCOMATRIX adjuvant to 5 �g of HA, after 2 doses, the serocon-ersion rate is 80.6% [16]. These results highlight the importancef having a potent adjuvant for use in future pandemics to ensureoverage with limited supplies of vaccine through antigen spar-ng effect as well as eliciting responses that are broad enough forrotection against antigenic variants.

cknowledgements

We thank Joseph Thong for his assistance in BSL3 work. Thisork is supported by funds from DSO National Laboratories andefence Research and Technology Office, Ministry of Defence,epublic of Singapore.

Contributors: LHT rescued various viruses used and performedarious in vitro assays. JHL and LFK performed the animal study.YFLesigned the experiments and performed the BSL3 works. LFK andFL drafted the manuscript.

Conflict of interest: All authors have no conflict of interest toeclare.

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