Homosubtypic and heterosubtypic antibodies against highly pathogenic avian influenza H5N1 recombinant proteins in H5N1 survivors and non-H5N1 subjects

Homosubtypic and heterosubtypic antibodies against highly pathogenic avian influenza H5N1 recombinant proteins in H5N1 survivors and non-H5N1 subjects

Virology 454-455 (2014) 254–262 Contents lists available at ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Homosubtypic and...

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Virology 454-455 (2014) 254–262

Contents lists available at ScienceDirect

Virology journal homepage: www.elsevier.com/locate/yviro

Homosubtypic and heterosubtypic antibodies against highly pathogenic avian influenza H5N1 recombinant proteins in H5N1 survivors and non-H5N1 subjects Pirom Noisumdaeng a,b, Phisanu Pooruk a, Jarunee Prasertsopon a, Susan Assanasen c, Rungrueng Kitphati d, Prasert Auewarakul a,b, Pilaipan Puthavathana a,b,n a Siriraj Influenza Cooperative Research Center, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok 10700, Thailand b Center for Emerging and Neglected Infectious Disease, Mahidol University, Nakhon Pathom 73170, Thailand c Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok 10700, Thailand d Department of Disease Control, Ministry of Public Health, Nonthaburi 11000, Thailand

art ic l e i nf o

a b s t r a c t

Article history: Received 13 December 2013 Returned to author for revisions 3 January 2014 Accepted 22 February 2014 Available online 21 March 2014

Six recombinant vaccinia viruses containing HA, NA, NP, M or NS gene insert derived from a highly pathogenic avian influenza H5N1 virus, and the recombinant vaccinia virus harboring plasmid backbone as the virus control were constructed. The recombinant proteins were characterized for their expression and subcellular locations in TK  cells. Antibodies to the five recombinant proteins were detected in all 13 sequential serum samples collected from four H5N1 survivors during four years of follow-up; and those directed to rVac-H5 HA and rVac-NA proteins were found in higher titers than those directed to the internal proteins as revealed by indirect immunofluorescence assay. Although all 28 non-H5N1 subjects had no neutralizing antibodies against H5N1 virus, they did have cross-reactive antibodies to those five recombinant proteins. A significant increase in cross-reactive antibody titer to rVac-H5 HA and rVac-NA was found in paired blood samples from patients infected with the 2009 pandemic virus. & 2014 Elsevier Inc. All rights reserved.

Keywords: Highly pathogenic avian influenza (HPAI) H5N1 virus Recombinant vaccinia virus Influenza recombinant proteins Homosubtypic antibodies Heterosubtypic antibodies Immunofluorescence assay Western blot assay

Introduction Based on antigenicity of hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are classified into 18 HA (H1–H18) and 11 NA (N1–N11) subtypes, respectively (Forrest and Webster, 2010; Tong et al., 2013; Zhu et al., 2013). All influenza A viruses belonging to subtypes H1–H16 are found in aquatic birds, but the subtypes H17 and H18 were discovered in bats. The influenza A subtypes seasonally circulating in human population include the H3N2 and 2009 pandemic H1N1 (H1N1pdm) viruses (Forrest and Webster, 2010; World Health Organization (WHO), 2013). Occasionally, certain avian influenza A viruses have crossed species barrier to infect humans for example: H5N1, H7N7, H7N2, H7N3 and H9N2 viruses (Centers for Disease Control and Prevention (CDC), 2013; Forrest and Webster, 2010; Peiris et al., 2007), and a

n Corresponding author at: Mahidol University, Faculty of Medicine Siriraj Hospital, Department of Microbiology, 2 Prannok Road, Bangkok 10700, Thailand. Tel.: þ 662 419 7059; fax: þ 662 418 2663. E-mail addresses: [email protected], [email protected] (P. Puthavathana).

http://dx.doi.org/10.1016/j.virol.2014.02.024 0042-6822 & 2014 Elsevier Inc. All rights reserved.

novel subtype H7N9 found in China in 2013 (Gao et al., 2013). The highly pathogenic avian influenza (HPAI) H5N1 virus is the most virulent avian virus ever reported to infect man. The genome of influenza A virus comprises 8 RNA segments which encode for 10–13 different proteins including 3 polymerase proteins: polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid protein (PA), nucleoprotein (NP), 2 matrix proteins: M1 and M2, and 2 nonstructural proteins: NS1 and NS2, and 2 surface glycoproteins: HA and NA (Das et al., 2010). In addition, PB1-F2 and N40 proteins which are translated from PB1 mRNA, and PA-X which is translated from PA mRNA, have been discovered in some influenza subtypes (Jagger et al., 2012; Wise et al., 2009). It is generalized that HA and NA proteins are highly variable and immunogenic; while the internal proteins (PB2, PB1, PA, NP, M and NS) are more conserved across influenza subtypes. During influenza virus infection, antibodies to all major viral proteins including surface glycoproteins and internal proteins are produced, indicating that these antigens are exposed to the humoral immune system (Kreijtz et al., 2011; Lynch et al., 2008; Sandbulte et al., 2007; Zhang et al., 2011). Antibodies to HA are the most important for prevention of illness. They can neutralize the

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viral infectivity by interfering with the viral attachment to host cell receptors, but they are relatively strain specific because the epitope responsible for the viral attachment is located on the HA1 variable domain. On the other hand, the antibody to HA2 which is the conserved domain of HA protein has been shown to exhibit cross-reactive neutralizing antibody (Sui et al., 2009; Corti et al., 2010). As the HA2 domain mediates the fusion between the viral envelope and endosomal membrane during the uncoating step of the replication cycle, therefore, it is assumed that this broadly neutralizing antibody inhibits virus uncoating, but not virus attachment. However, antibodies against the other conserved proteins also mediated anti-viral activity through various mechanisms (Carragher et al., 2008; El Bakkouri et al., 2011; Jegaskanda et al., 2013; Jegerlehner et al., 2004; LaMere et al., 2011; Mozdzanowska et al., 1999; Staneková and Varečková, 2010). As the effectiveness of influenza vaccine is limited by the emerging of drifted HA or novel HA subtypes, the development of vaccine with broader reactivity is crucial. Basically, this type of vaccine should target the conserved proteins such as those internal proteins. Nevertheless, the information on magnitude of the antibody response to single viral protein is limited owing to lack of the test antigen. In another word, it is not easy to demonstrate the subtype-specific and cross-reactive antibody against individual influenza protein of interest without interference from the other influenza proteins. Vaccinia virus has been widely used as a powerful expression vector for production of antigenically and biologically active proteins (Hruby, 1990; Mackett and Smith, 1986). Several seasonal influenza proteins, including PB2, PB1, PA, HA, NA, NP, M and NS derived from A/PR/8/34 (H1N1) have been expressed in the recombinant vaccinia virus infected cells and used to demonstrate the influenza subtype-specific and cross-reactive cellular immunity in mice and humans (Andrew et al., 1986; Jameson et al., 1998). In the present study, five recombinant vaccinia viruses harboring HPAI H5N1 HA, NA, NP, M or NS gene insert were constructed. The thymidine kinase negative (TK  ) cells infected with these viruses were used as the test antigens for detection of antibodies to recombinant proteins in H5N1 survivors and nonH5N1 subjects by immunofluorescence (IF) assay. The data on homosubtypic and heterosubtypic antibodies against an individual H5N1 protein in our subjects provided a choice of target proteins

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for universal vaccine development as well as broaden the understanding of influenza immunity.

Results Characterization of the H5N1 recombinant proteins Investigation on the expression and characterization of the H5N1 recombinant proteins were performed by WB and IF assays. WB assay demonstrated 3 forms of rVac-H5 HA, the uncleaved HA0 precursor and its HA1 and HA2 cleavage products whose molecular weight (MW) were comparable to those of the native HA proteins synthesized in the MDCK cells infected with A/Thailand/1(KAN-1)/2004 (H5N1) (KAN-1 virus), i.e., 75, 55 and 27 kDa, respectively (Fig. 1, left panel). The rVac-NA, rVac-NP, rVac-M and rVac-NS proteins had MW of 50, 56, 27 and 26 kDa, respectively; these were also similar to those synthesized in the KAN-1 virus infected MDCK cells (Fig. 1, right panel). Confocal microscopy showed that the rVac-H5 HA and rVac-NA proteins localized both in the cytoplasm, and in particular, on the cytoplasmic membrane. The rVac-M protein was mainly found in the cytoplasm; while the rVac-NP and rVac-NS proteins were found both in the cytoplasm and nucleus. The TK  cells infected with rVac-pSC11 virus control did not show any fluorescent positive signal (Fig. 2). Detection of antibodies against H5N1 viral proteins by IF assay IF assay was employed for detection of antibodies against rVacHA, rVac-NA, rVac-NP, rVac-M and rVac-NS in 13 sequential serum samples from 4 H5N1 survivors and 38 serum samples from nonH5N1 subjects (paired blood samples from 5 patients infected with 2009 pandemic A(H1N1) (H1N1pdm) influenza virus and 5 patients infected with H3N2 influenza virus and single blood samples from 18 healthy individuals). In parallel, TK  cells infected with rVac-pSC11 virus were used as the control antigen. The results demonstrated that all serum samples from H5N1 survivors had antibodies to the 5 H5N1 recombinant proteins with the titers within the range of 40 to 640; all of these sera had the antibody titer of 20 against the recombinant control antigen.

Fig. 1. Expression of rVac-H5 HA, rVac-NP, rVac-NA, rVac-M and rVac-NS proteins in TK  cells infected with recombinant vaccinia virus as demonstrated by WB assay. The molecular weight of these recombinant proteins is similar to those native proteins synthesized in MDCK cells infected with KAN-1 virus, but it is higher than those expressed by the baculovirus-insect cell system (rBV-H5 HA). The HA0, HA1 and HA2 domains at the molecular weight of 75, 55 and 27 kDa are demonstrated in the left panel and the NP, NA, M1 and NS1 at the molecular weight of 50, 56, 27 and 26 kDa, respectively are demonstrated in the right panel. The asterisk (n) is the recombinant vaccinia virus expressing NP, NA, M or NS.

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Fig. 2. IF assay for expression and localization of H5N1 recombinant proteins in the infected TK  cells as visualized under a confocal microscope at objective magnification of 63  with zoom 2  . TK  cells infected with rVac-pSC11 virus is used as the negative control.

The antibody titers to rVac-H5 HA and rVac-NA proteins were markedly higher than those to the internal proteins: rVac-NP, rVac-M and rVac-NS. Additionally, those antibodies could last for

longer than 3 or 4 years as we could follow up that far (Table 1). The result from paired blood samples of all 5 H1N1pdm patients showed a significant increase in geometric mean titer (GMT) of

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Table 1 Homosubtypic antibodies against HPAI H5N1 viral proteins in H5N1 survivors as determined by IF assay. Survivor no.

Age (years)

Blood sample

Time at blood collection after disease onset

NT antibody titer to

IF antibody titer to

H1N1

H3N2

H5N1

HA

NA

NP

M

NS

Control antigena

1

32

1 2 3

1y 6m 2y 6m 3y 8m

ND 640 ND

ND 10 ND

160 160 80

640 640 640

320 320 160

80 80 160

160 80 80

160 160 160

20 20 20

2

29

1 2 3

2y 2m 3y 2m 4y 3m

ND 20 ND

ND 80 ND

160 160 80

320 160 320

160 320 160

80 80 80

40 40 40

40 160 80

20 20 20

3

7

1 2 3 4

20d 11m 2y 2m 3y 1m

ND 320 ND ND

ND o 10 ND ND

640 80 80 40

320 320 160 160

320 160 80 80

320 160 160 80

80 80 80 40

160 160 160 160

20 20 20 20

4

2

1 2 3

2y 3m 3y 11m 4y 11m

ND 160 ND

ND o 10 ND

80 80 40

80 80 80

80 160 160

40 40 40

40 40 40

40 80 40

20 20 20

y, year; m, month; d, day; NT Ab, neutralizing antibody; IF Ab, immunofluorescence antibody. ND, not determine. H1N1: A/Thailand/Siriraj-Rama-TT/2004 (H1N1), an A/New Caledonia/20/1999 (H1N1)-like. H3N2: A/Siriraj ICRC/SI-154/2008 (H3N2), an A/Brisbane/10/2007 (H3N2)-like virus. H5N1: A/Thailand/1(KAN-1)/2004 (H5N1) virus. a

Control antigen is prepared from TK- cells infected with rVac-pSC11 virus.

antibodies against the rVac-H5 HA and rVac-NA surface glycoproteins, but not to the other recombinant internal proteins (Wilcoxon Signed Ranks test, p o0.05). In contrast, no significant increase in antibody titers against any of the 5 recombinant proteins was found in paired sera from all 5 H3N2 patients. All 18 healthy subjects also contained the cross-reactive antibodies against the 5 H5N1 recombinant viral proteins at various titers (Fig. 3A–E). Additionally, 28 of all 32 subjects contained low antibody titers (GMT of 13.4) to vaccinia virus when rVac-pSC11 virus infected cells was used as the tested antigen (Fig. 3F).

Detection of neutralizing antibodies against influenza A viruses An ELISA-based microneutralization (microNT) assay was employed for detection of neutralizing antibodies against H1N1, H3N2 and HPAI H5N1 viruses. All of 4 H5N1 survivors contained neutralizing antibodies to both the seasonal H1N1 and H3N2 viruses and markedly high antibody titers against H5N1 virus (Table 1). In contrast, the non-H5N1 subjects had no neutralizing antibody against H5N1 virus as screen at the serum dilution of 1:10; while they possessed antibodies directed against H1N1 and/ or H3N2 viruses as shown in Table 2.

Conservancy of amino acid sequences among various influenza subtypes Amino acid sequences of KAN-1 virus and human influenza A subtypes H1N1, H3N2 and H1N1pdm were retrieved from the GenBank database and aligned to each other using clustalW multiple alignment. Accession numbers of these viruses are shown in the supplementary Table S1. The percentages of amino acid identity were determined by the sequence identity matrix application in BioEdit program version 7.0.4.1, and showed the identity of more than 90% for PB2, PB1, PA, NP and M1, and 76.5–91.7% for M2, NS1 and NS2. Much less identity was found in HA and NA proteins (Table 3).

Discussion Herein, the recombinant vaccinia virus harboring HA, NA, NP, M or NS gene insert derived from HPAI H5N1 KAN-1 virus was constructed; the subcellular location of the expressed proteins was then demonstrated by IF assay using confocal microscope. The results were similar to those previous reports such that the rVac-H5 HA and rVac-NA proteins localized in the cytoplasm and particular on the cytoplasmic membrane; and the rVac-M, rVac-NP and rVac-NS proteins were found both in the cytoplasm and nucleus of the infected cells (Smith et al., 1987). The MW of the three forms of rVac-H5 HA in this study was similar to those previously reported in the other studies and our previous lot of recombinant protein, i.e., 75 or 80 for HA0, 55 or 58 for HA1 and 25 or 27 kDa for HA2 (Noisumdaeng, et al., 2013; Privalsky and Penhoet, 1978; Skehel and Waterfield, 1975). Our WB assay also demonstrated the similarity in MW between rVac-NA, rVac-NP, rVac-M or rVac-NS and those native proteins synthesized in the KAN-1 virus infected MDCK cells. Collectively, the posttranslational modification process such as glycosylation in HA and NA (Gallagher et al., 1992; Markoff et al., 1984) and phosphorylation in NP, M and NS (Gregoriades et al., 1990; Privalsky and Penhoet, 1978) in vertebrate cells, i.e., TK  cells from human origin and MDCK from dog origin should be similar. The MW of 50, 56, 27 and 26 corresponding to our rVac-NA, rVac-NP, rVac-M and rVac-NS, respectively was similar to those reported in the other studies, i.e., 45 or 58 kDa for NA, 56 or 60 kDa for NP, 26 or 27 kDa for M1, and 25 kDa for NS1 (Klenk and Rott, 1973; Privalsky and Penhoet, 1978; Shaw et al., 2008). However, our study did not find the splicing of M mRNAs for M2 protein or NS mRNAs for NS2 protein, because both M2 and NS2 proteins were not demonstrated by our WB assay using specific monoclonal antibody to M2 and NS2 proteins, respectively. The absence of splicing of M and NS mRNAs in the recombinant vaccinia virus expression system was also noted by the other investigators (Smith et al., 1987). Therefore, it is likely that the rVac-M and rVac-NS proteins expressed in our system might be M1 and NS1 according to their MW and reactivity with the specific antibodies to M1 and NS1, respectively. We employed TK  cells infected with recombinant vaccinia virus as the test antigen to detect homosubtypic and heterosubtypic

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Fig. 3. Determination for cross-reactive non-neutralizing antibodies against the recombinant H5N1 viral proteins in non-H5N1 subjects by IF assay: HA (A), NA (B), NP (C), M (D) and NS (E), and the rVac-pSC11 virus infected TK- cells as control antigen (F). Only H1N1pdm patients show a significant increase in GMT against HA and NA proteins in paired sera (Wilcoxon Signed Ranks test, p o0.05).

antibodies to H5N1 HA, NA, NP, M and NS proteins in 4 survivors of H5N1 infection and 28 non-H5N1 subjects by IF assay. Our limitation was the number of subjects participated in this study. There are 25 laboratory confirmed cases of H5N1 infection with 17 deaths in Thailand, and we could enroll only half of the survivors. Nevertheless, it is laborious and not practical for grading the antibody titer in large sample size by IF assay. We preliminarily aimed to demonstrate the existing of cross-reactive immunity to H5N1 proteins in non-H5N1 subjects; and enrollment of 28 nonH5N1 subjects revealed presence of such antibodies in all subjects, and to all kinds of H5N1 proteins. In H5N1 survivors, the antibody titers to the rVac-H5 HA and rVac-NA were markedly higher than those to the internal proteins, suggesting that the surface glycoproteins HA and NA are more immunogenic. Additionally, those antibodies could last for 3 to 4 years and could be followed up that for that long.

The heterosubtypic antibodies against those 5 H5N1 recombinant proteins were demonstrated by IF assay in all 28 non-H5N1 subjects who had no neutralizing antibody to H5N1 virus. A fourfolded rise in antibody titer against HA, NA, or HA and NA or NP was found in paired sera from all 5 H1N1pdm patients, but with a significant increase of GMT to HA and NA only. In contrast, a significant increase in cross-reactive antibody against any of the H5N1 viral proteins was not found in the H3N2 patients. These findings could be explained such that the HA of H1N1 and H5N1 viruses belonged to the same phylogenetic clade (Medina and Garcĭa-Sastre, 2011). The H1N1pdm virus and the H5N1 virus were 62% and 84% identical in HA and NA amino acid sequences, respectively; while the HA and NA of H3N2 virus is quite distinct from the H5N1 virus by showing only 39% identity in HA and 41% in NA. Thus, it is postulated that the patients recently infected with H1N1pdm virus could generate stronger cross-reactive antibodies

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Table 2 NT antibody titers obtained from the 28 non-H5N1 subjects. Virus subtype

H1N1a

Subject group

Blood sample

H1N1pdm patients (n¼5) H3N2 patients (n¼5)

H3N2

Healthy (n ¼18) H1N1pdm patients (n¼5) H3N2 patients (n¼5)

H5N1

Healthy (n ¼18) H1N1pdm patients (n¼5) H3N2 patients (n¼5) Healthy (n ¼18)

Number of cases at NT antibody of

Acute Conv. Acute Conv. Single Acute Conv. Acute Conv. Single Acute Conv. Acute Conv. Single

o10

10

20

40

1

2

1

1

3 1

4 2 5 5 18

1 1 3

1 2

1 1

2

GMT 41280

80

160

320

640

2 2 2 3

1

1 1 2

2 1 1 1

4

1 1 8

1 3

2

6

5

1 2

2 1

2 1

13.2 278.6 367.6 367.6 718.4 15.2 26.4 105.6 970.1 141.1 5.7 7.6 5 5 5

H3N2: A/Siriraj ICRC/SI-154/2008 (H3N2), an A/Brisbane/10/2007 (H3N2)-like virus. H5N1: A/Thailand/1(KAN-1)/2004 (H5N1) virus. Conv., Convalescent. a H1N1: A/Thailand/Siriraj-Rama-TT/2004 (H1N1), an A/New Caledonia/20/1999 (H1N1)-like virus is used as the test virus in H3N2 patients and healthy individuals; while A/Thailand/104/2009 (H1N1pdm) virus is used as the test virus in the H1N1pdm patients.

Table 3 Percentages of amino acid sequence identities among various influenza subtypes. Protein

Amino acid conservancy rate (% identity)a Seasonal H1N1

H5N1 (KAN-1)

PB2 PB1 PA whole HA HA1 HA2 NP NA M1 M2 NS1 NS2

Seasonal H3N2

2009 H1N1pdm

New Caledonia/20

Brisbane/59

Brisbane/10

Wisconsin/67

California/07

Thailand/104

95.1 95.1 94.2 62.6 51.1 80.6 92.3 79.7 92.4 81.4 83.4 89.2

94.9 95.3 94.1 62.1 50.8 79.7 92.3 78.9 93.2 80.4 82.6 88.4

94.4 96.6 93.8 39.5 33.3 49.5 90.9 41.4 94 84.5 79.5 90.9

94.5 96.4 94.1 38.8 32.7 48.6 91.5 41 94 84.5 79.5 90.9

97.1 96 96.2 62.2 50 81.5 93.3 84.2 96 91.7 76.5 87.6

96.8 95.9 96.2 62.2 50.2 81 93.7 84.2 96 91.7 76.5 87.6

a Influenza viruses include A/Thailand/1(KAN-1)/2004 (H5N1), A/New Caledonia/20/1999 (H1N1), A/Brisbrane/59/2007 (H1N1), A/Brisbane/10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2), A/California/07/2009 (H1N1) and A/Thailand/104/2009 (H1N1). Each protein of all influenza viruses is aligned and analyzed for the percentages of amino acid identity by using sequence identity matrix in BioEdit program.

against the conserved epitopes in HA and NA than those infected with H3N2 virus. Using ELISA, previous investigators demonstrated that individuals infected with seasonal viruses developed cross-reactive antibodies against HA antigen derived from different strains and even different subtypes, i.e., avian H8 HA (Burlington et al., 1985) and avian H5 HA (Stelzer-Braid et al., 2008). Our previous work also demonstrated the presence of antibody to H5 HA2 domain in all non-H5N1 subjects by WB assay (Noisumdaeng et al., 2013). The H5 HA2 domain has also been shown by various groups of investigators for its broad antigenicity and capability to induce cross-reactive neutralizing antibody (Sui et al., 2009; Corti et al., 2010). Moreover, previous investigators also reported low neutralizing antibody activities (Zhang et al., 2011) or cross-reactive binding antibodies against H5N1 proteins by WB assay in general population (Lynch et al., 2008). Our previous report also demonstrated cross neutralizing antibody to H5N1 KAN-1 virus in vaccinees who received seasonal influenza vaccine (Kositanont et al., 2010).

It is noted that our H5N1 survivors and non-H5N1 subjects contained low level of antibody to the control antigen (rVac-pSC11 infected cells) in IF assay. Of all 51 sera including 13 sequential serum samples from 4 H5N1 survivors and 38 serum samples from 28 non-H5N1 subjects, the GMT of 14.8 were found. Thailand stopped smallpox vaccination in 1980; therefore, it cannot be ruled out that the antibody to the control antigen found in the subjects of age older than 33 years might be due to the long last persisting anti-vaccinia virus antibody. In particular, the two cases with the titer of 40 were 43 and 56 years old. At presence, it is still not known why the non-vaccinia immunized subjects contained the background of antibody to the control antigen. The non-neutralizing antibodies might exhibit the important role in the protection against influenza virus infection. Antibodies against HA, NA and M2 facilitated antibody dependent cellular cytotoxicity which contributed to the clearance of the virus infected cells (Kreijtz et al., 2011; Jegaskanda et al., 2013; Jegerlehner et al., 2004; Mozdzanowska et al., 1999). The antibodies to M2e

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and NP could eliminate the virus by FcR-mediated phagocytosis (El Bakkouri et al., 2011; LaMere et al., 2011). Passive transfer of immune serum obtained from donor mice immunized with the recombinant NP could promote virus clearance in the recipient mice after challenging with influenza H1N1 or H3N2 viruses (LaMere et al., 2011). Moreover, the anti-NP antibodies could promote dendritic cell maturation, Th1 cytokine production and, in addition, stimulate complement-mediated lysis of the influenza virus infected P815 cells (Yewdell et al., 1981; Zheng et al., 2007). Based on these data, it is clear that non-neutralizing antibodies against internal conserved proteins also confer some degree of protection. Our findings, together with those earlier reports, showed that the cross-reactive neutralizing and non-neutralizing antibodies against the conserved epitopes of H5N1 viral proteins exist in general population, probably as a result of previous infection in young lives or annual influenza vaccination. As the antibody against HA is relatively strain specific, the effectiveness of annual vaccine is limited by its inability to induce the antibody that can neutralize an emerging variant virus. Currently, the influenza universal vaccines are targeting on the conserved epitopes of HA and M proteins based on their functions. The other influenza viral proteins such as NA and NP could also confer some effective antiinfluenza viral activities in vitro and in vivo. However, it is to be further explored if the antibodies to conserved epitopes in other influenza viral proteins as determined by our IF assay could confer effective anti-viral activities.

Materials and methods Ethical issues This study has been approved by two Institutional Review Boards; the first one from the Faculty of Medicine Siriraj Hospital, Mahidol University, and the second one from the Ministry of Public Health, Thailand. Subjects signed in the consent form for participation in this study. Human subjects and blood specimen collection The anonymous sera in this study comprised 13 sequentially archived serum samples from 4 H5N1 survivors, 10 paired sera collected at approximately 2–3 weeks apart from 5 H1N1pdm patients and 5 H3N2 patients, and 18 single serum samples from healthy individuals. Ages of the H5N1 survivors are shown in Table 1. Mean ages (age-ranges) of the non-H5N1 subjects were as follows: 21 (21) for H1N1pdm patients; 25 (24–27) for H3N2 patients and 34 (23–56) for healthy subjects. All patients were diagnosed by real time reverse transcription polymerase chain reaction (real time RT-PCR), virus isolation and/or serodiagnosis. Serum samples were kept frozen at  20 1C until used. The study viruses Influenza viruses employed in the present study included HPAI H5N1 virus, A/Thailand/1(KAN-1)/2004 (H5N1) clade 1 (KAN-1 virus), A/Thailand/Siriraj-Rama-TT/2004 [A/New Caledonia/20/1999 (H1N1)-like virus], A/Thailand/104/2009 (H1N1) virus (H1N1pdm virus), and A/Siriraj ICRC/SI-154/2008 [A/Brisbane/10/2007 (H3N2)like virus]. These viruses were propagated in Madin-Darby canine kidney (MDCK) cells maintained in Earle's minimal essential medium (EMEM) (Gibco, Carlsbad, CA) in presence of trypsin-tosyl phenylalanyl chloromethyl ketone (trypsin-TPCK) (Sigma-Aldrich, St. Louis, MO) and antibiotics, and without fetal bovine serum (FBS) (Gibco) supplement.

Vaccinia vaccine virus strain Lister, kindly provided by the Thai Government Pharmaceutical Organization, was used as the parental virus for construction of the recombinant viruses harboring HPAI H5N1 HA, NA, NP, M or NS gene insert. These vaccinia viruses were propagated in TK  cells maintained in Dulbecco's modified eagle medium (DMEM) (Gibco) supplemented with 10% FBS. The virus stocks were kept at 80 1C until used. Construction of recombinant vaccinia viruses The steps of the construction of recombinant vaccinia viruses involved cloning of H5N1 gene of interest into pGEMs-T Easy plasmid (Promega Corporation, Madison, WI) and subcloning into pSC11 vector before transfection into TK  cells pre-infected with the vaccinia virus vaccine strain. Total RNA was extracted from MDCK cells infected with KAN-1 virus using a QIAamps viral RNA mini kit (Qiagen, GmbH, Hilden, Germany). The complete genomic segment including HA, NA, NP, M and NS was amplified by OneStep RT-PCR kit (Qiagen) using universal primers (Hoffmann et al., 2001). The HA, NA, NP, M and NS PCR products at size of 1,807, 1,429, 1,594, 1,056 and 905 base pairs (bp) in length, respectively were gel electrophoresed and purified by using QIAquicks gel extraction kit (Qiagen) and cloned into pGEMs-T Easy vector by using T4 DNA ligase (Promega) before transforming into Escherichia coli JM109 cells. The recombinant plasmids extracted from the recombinant bacteria were digested with NotI enzyme to yield the sticky end products that were further repaired by Klenow DNA polymerase (New England Biolabs Inc., Ipswich, MA) in order to generate blunt end DNA strands. The DNA product was subcloned into a pSC11 expression vector kindly provided by Prof. Bernard Moss, the National Institute of Allergy and Infectious Disease, Maryland, USA. This vector contains the SmaI insertion site located downstream of vaccinia virus p7.5 promoter together with the E. coli lacZ gene which encodes for β-galactosidase under a p11 promoter, and is flanked with thymidine kinase sequences (TKR and TKL). E. coli were transformed with the recombinant plasmids and plated on Luria-Bertani (LB) agar containing 5-bromo-4-chloro-3-indolyl-βD-galactopyranoside (X-gal) (Promega) plus ampicillin as the selective marker. The cloned bacterial colonies were cultured in LB broth and the plasmids were extracted by a QIApreps Spin mini kit (Qiagen). The presence of H5N1 gene insert with correct orientation was investigated by cutting the purified recombinant plasmids by restriction enzymes XhoI/PstI, BamHI/XhoI, PstI/XhoI, XhoI and HindIII/XhoI (New England Biolabs) for pSC11-H5 HA, pSC11-NA, pSC11-NP, pSC11-M and pSC11-NS, respectively. DNA sequencing was performed in order to determine the in-frame translation of the H5N1 gene insert (Sequencing primers available on request). To construct a recombinant vaccinia virus containing H5 HA, NA, NP, M or NS gene insert (rVac-HA, rVac-NA, rVac-NP, rVac-M or rVac-NS, respectively), a mixture of pSC11 recombinant plasmid together with DMRIE-C transfection reagent (Invitrogen, Carlsbad, CA) in DMEM was inoculated onto the TK  cell monolayer that were pre-infected with parental vaccinia vaccine virus at the multiplicity of infection (MOI) of 0.01 plaque forming unit (PFU)/ml for 2 h. The H5N1 DNA flanked with TKR and TKL was inserted into the parental vaccinia viral genome by homologous recombination with the tk gene. The transfected culture was further incubated for 2 days to allow virus replication. As a result, the recombinant virus harboring the H5N1 gene insert gains the TK  phenotype, which results in the loss of the ability to produce thymidine kinase enzyme. The recombinant vaccinia virus was distinguished from the parental TK þ vaccinia virus by plaque selection on the TK  cell monolayer maintained in low melting point agarose containing 5-bromo-2'-deoxyuridine (BrdU) (Sigma

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Aldrich, St. Loius, MO) and X-gal, in which the plaques produced by cells infected with the recombinant vaccinia virus appeared blue. Plaque purification was performed three times in order to obtain the single clone of the recombinant virus. In parallel, the recombinant vaccinia virus containing pSC11 vector (rVac-pSC11) was constructed for using as the vaccinia virus control. The recombinant vaccinia virus was propagated and titrated in TK  cells by plaque assay, and further characterized by WB and IF assays. Preparation of H5N1 viral antigens TK  cells infected with the recombinant vaccinia virus harboring H5N1 HA, NA, NP, M or NS gene insert were used as the antigens in IF and WB assays. Additionally, MDCK cells infected with KAN-1 virus was used as the positive control antigen. For WB assay, the infected cell pellets were re-suspended with RIPA buffer (50 mM Tris Cl pH 7.5, 150 mM NaCl, 1% Tritons X-100, 0.5% sodium deoxycholate, 0.1% SDS) to yield the cell lysates containing the test antigens. For IF assay, the infected cell suspensions were deposited on microscopic glass slides, air dried and fixed with precooled acetone at  20 1C for 10 min. The fixed slides were kept at 80 1C until stained. Western blot assay HA, NA, NP, M and NS proteins expressed in TK  cells infected with the recombinant vaccinia virus were characterized in comparison with the native H5N1 viral proteins produced in MDCK cells infected with H5N1 KAN-1 virus by WB assay. Briefly, the infected cell lysates or recombinant antigens were mixed with 4X reducing sample buffer (8% SDS, 250 mM Tris Cl pH 6.8, 8% β-mercaptoethanol, 0.4% bromophenol blue, 40% glycerol), boiled at 95 1C for 5 min and subjected to 10% SDS-PAGE. The proteins in gel were blotted onto a nitrocellulose membrane (Protrans, Whatman, GmbH, Germany) by using Trans-Blots SD semidry transfer cell (Bio-Rad). The blotted membrane was blocked with 5% skim milk in Tris-buffer saline plus 0.1% tween-20 (TBS-T). MW of the native and recombinant H5N1 viral proteins was determined using the specific primary antibodies as follows: goat antiserum against A/Vietnam/1203/2004 (H5N1) (VN1203) HA kindly provided by Prof. Robert G. Webster and Dr. Richard Webby, St. Jude Children's Research Hospital, TN; mouse monoclonal antibody to VN1203 HA as well as rabbit polyclonal antibody against synthetic peptide corresponding to 15 amino acids at the carboxy terminus of NA from avian influenza H5N1 virus (US Biological, Swampscott, MA); goat antibody to M protein (US Biological, Swampscott, MA); mouse monoclonal antibody to NP (Millipore Corporation, Temecula, CA); and goat anti-NS1 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The anti-species specific immunoglobulin comprising horseradish peroxidase (HRP) enzyme conjugated rabbit anti-goat Igs (Dako Cytomation, Glostrup, Denmark), goat anti-rabbit Igs (Zymed Laboratories, San Francisco, CA) or goat anti-mouse Igs (Dako) was used as the secondary antibody. The blotted membrane was incubated with the primary antibody overnight at 41C before washing with TBS-T and followed by incubation with the corresponding secondary antibody for 2 h at room temperature. The mixture of 3,3'-diaminobenzidine (Sigma-Aldrich, St. Louis, MO), 8% NiCl2 and 6% H2O2 was used as the chromogenic substrate.

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purpose was to determine the antibody titers against H5N1 proteins in human sera. Cell deposits were incubated with primary specific antibodies for 1 h at 37 1C, and then followed by the secondary antibodies: fluorescein isothiocyanate (FITC) conjugated rabbit anti-goat Igs (Dako Cytomation), goat anti-rabbit Igs (Abcam, plc., Cambridge, UK), goat anti-mouse Igs (Light Diagnostics™, Temecula, CA) or goat anti-human IgG (Invitrogen, Frederick, MD) for 1 h at 37 1C and counter stained with 0.5% Evan's blue dye. The stained slides were examined under an ordinary fluorescence microscope (Nikon Eclipse 80i, Japan) by two independent scientists in order to avoid bias in grading the antibody titer. The reciprocal of the last serum dilution which yielded the fluorescence intensity of 2 þ was considered to be the antibody titer. On the other hand, the stained slides for confocal microscopy were counter stained with trihydrochloride trihydrate (Hoechst 33342Invitrogen, Eugene, Oregon) for nuclear staining together with 0.5% Evan's blue dye for 10 min. The slides were examined for fluorescent cells under a laser scanning confocal microscope (LSM 510 Meta, Zeiss, Jena, Germany). Microneutralization assay ELISA-based microNT assay was carried out in duplicate in MDCK cell monolayers. The protocol was conducted according to that described previously (Lerdsamran et al., 2011). Briefly, the test serum was treated with receptor destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) and followed by heat inactivation at 56 1C for 30 min. The treated serum was serially 2 fold-diluted before adding with the test virus at final concentration of 100 TCID50/ reaction in duplicate. Amount of viral nucleoprotein in the reaction wells was determined by ELISA using mouse-specific anti-NP monoclonal antibody (Chemicon, Temecula, CA) as the primary antibody, and HRP conjugated goat anti-mouse Igs (Southern Biotechnology, Birmingham, AL) as the secondary antibody, and TMB (KPL Inc., Gaithersburg, MD) as the chromogenic substrate. The neutralizing (NT) antibody titer was defined as the reciprocal of the highest serum dilution that reduces Z50% of the amount of viral nucleoprotein in the reaction wells as compared to the virus control wells. The geometric mean titer (GMT) was calculated by assigning the antibody titer o 10 as 5. Acknowledgments This work was supported by the Thailand Research Fund for Senior Research Scholar, the National Science and Technology Development Agency, and the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative. We thank our subjects for providing blood samples; and the Division of Medical Molecular Biology, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University for the laser scanning confocal microscope facilities.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.02.024. References

Immunofluorescence assay IF assay was performed for 2 purposes in this study. The first one was to investigate for the expression and localization of the H5N1 recombinant proteins expressed in TK  cells. The second

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