Since the Hong Kong pandemic of 1968, H3N2 influenza A

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1 H3N2 Influenza Virus Infection Induces Broadly Reactive Hemagglutinin Stalk Antibodies in Humans and Mice Irina Margine, a,b Rong Hai, a Randy A. Albrecht, a,c Gerlinde Obermoser, d A. Carson Harrod, d Jacques Banchereau, d Karolina Palucka, d Adolfo García-Sastre, a,c,e Peter Palese, a,e John J. Treanor, f Florian Krammer a Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA a ; Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA b ; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA c ; Baylor Institute for Immunology Research, Dallas, Texas, USA d ; Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA e ; Department of Medicine, University of Rochester Medical Center, Rochester, New York, USA f Broadly neutralizing antibodies directed against the conserved stalk domain of the viral hemagglutinin have attracted increasing attention in recent years. However, only a limited number of stalk antibodies directed against group 2 influenza hemagglutinins have been isolated so far. Also, little is known about the general level of induction of these antibodies by influenza virus vaccination or infection. To characterize the anti-stalk humoral response in the mouse model as well as in humans, chimeric hemagglutinin constructs previously developed in our group were employed in serological assays. Whereas influenza virus infection induced high titers of stalk-reactive antibodies, immunization with inactivated influenza virus vaccines failed to do so in the mouse model. Analysis of serum samples collected from human individuals who were infected by influenza viruses also revealed the induction of stalk-reactive antibodies. Finally, we show that the hemagglutinin stalk-directed antibodies induced in mice and humans have broad reactivity and neutralizing activity in vitro and in vivo. The results of the study point toward the existence of highly conserved epitopes in the stalk domains of group 2 hemagglutinins, which can be targeted for the development of a universal influenza virus vaccine in humans. Since the Hong Kong pandemic of 1968, H3N2 influenza A viruses have continually circulated in humans. During this time, these strains have undergone constant genetic drift, with genetically similar viruses prevailing for one or two seasons before receding. H3N2 viruses tend to predominate most influenza seasonal epidemics (1), including the past one, with infections being mostly prevalent in children and the elderly (2, 3). Although they are generally associated with uncomplicated disease in young healthy adults, epidemics caused by H3N2 strains have generally been more severe than those caused by H1N1 or influenza B virus (4). Vaccines remain the most reliable method to control seasonal epidemics of influenza. They induce humoral responses which are mainly directed to the two surface glycoproteins, the hemagglutinin (HA), which binds sialic acid-bearing receptors and mediates virus entry, and the neuraminidase (NA), which cleaves the receptors to facilitate virus release. Current influenza virus vaccines predominantly induce anti-ha antibodies that specifically target antigenic sites in the globular head domain and block its receptorbinding function (5, 6). However, these responses are strain specific in that the antibodies have potent neutralizing activity against homologous strains but have very limited breadth of reactivity due to the high variability of these sites. Hence, the effectiveness of vaccination relies on the accuracy of the prediction of the vaccine strains. In most years, the process is successful, but drifted variants and mismatches between the circulating viruses and the selected vaccine strain have occurred in the past, with important consequences to disease burden (7 10). Recently, another class of antibodies directed against the highly conserved stalk domain of the HA has gained increasing attention. Stalk-reactive antibodies tend to exhibit a much broader reactivity and neutralizing activity than antibodies targeting the globular head domain. A growing collection of stalk-reactive antibodies have been isolated from mice and humans, with the vast majority neutralizing group 1 HA-expressing influenza viruses (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and H17) (11 15). In contrast, only three neutralizing antibodies targeting group 2 HA stalks (H3, H4, H7, H10, H14, and H15) have been described (16 18), one of which cross-reacts to members of both group 1 and 2 HA families (16). These antibodies were isolated either from humans who were exposed to influenza virus by infection or vaccination or from mice sequentially vaccinated with DNA vaccines coding for divergent HAs. However, the frequency of B cells with specificities for the stalk domain of group 2 HAs is overall low, and it is unlikely that protective titers are induced upon vaccination with inactivated influenza vaccines. Aside from the three monoclonal antibodies mentioned above, the humoral response to conserved epitopes that are potentially found in group 2 HA stalk domains remains poorly characterized, and there are no quantitative data in the literature analyzing the induction of these antibodies upon infection or vaccination. Chimeric HA constructs expressing a globular head domain from one influenza A virus subtype and a stalk region from another subtype have recently been developed to measure stalk-reactive antibodies (19). By making use of these novel reagents, we showed that stalk-specific antibodies are induced following productive H1N1 infection of mice and humans (20, 21). One broadly neutralizing group 2 stalk antibody was previously isolated from a Received 20 December 2012 Accepted 6 February 2013 Published ahead of print 13 February 2013 Address correspondence to Florian Krammer, florian.krammer@mssm.edu. Copyright 2013, American Society for Microbiology. All Rights Reserved. doi: /jvi jvi.asm.org Journal of Virology p April 2013 Volume 87 Number 8

2 Stalk-Reactive Antibodies Induced by H3N2 Infections volunteer who had received the seasonal vaccine (16), but the extent to which such antibodies are induced in humans remains unclear. This study was conducted to quantitatively characterize the polyclonal group 2 HA stalk-reactive antibody response induced by influenza vaccination or by productive influenza virus infection of mice and humans. We also assessed the breadth of these antibodies by determining the degree of heterosubtypic cross-reactivity to H4 and H7 HAs. We show for the first time that influenza virus infection in mice and humans stimulates polyclonal antibody responses directed against the conserved stalk region of group 2 HA proteins that are sufficient to inhibit viral replication in vitro. This observation is the first clear indication that the stalk region of the group 2 HAs contains functional epitopes which have the potential to be targeted by a universal vaccine for influenza viruses. MATERIALS AND METHODS Cells and viruses. Madin-Darby canine kidney cells (MDCK) (ATCC CCL-34) were grown in minimal essential medium (MEM) (Gibco), and 293T cells (ATCC CRL-11268) were grown in Dulbecco s modified Eagle s medium (DMEM) (Gibco), both supplemented with 10% fetal bovine serum (HyClone) and penicillin/streptomycin (Gibco) (100 units/ml and 100 g/ml, respectively). The chimeric H7/3N3 virus (ch7/3-h7 HA globular head domain from A/mallard/Alberta/24/2001 virus [H7N3] and H3 HA stalk domain from A/Perth/16/2009 virus [Perth09, H3N2]) was rescued as described previously (19). Rescue transfections included pdz plasmids encoding the ch7/3 HA, the N3 NA genomic segment from A/swine/Missouri/ /2006 (H3N2) virus, and the remaining segments from A/PR/8/ 1934 (H1N1) virus (19). For the rescue, 293T cells were transfected with 1 g of all eight plasmids using Lipofectamine 2000 (Invitrogen). Twentyfour hours posttransfection, cells were mechanically detached and the cell/medium mixture was transferred into 10-day-old embryonated chicken eggs. Allantoic fluid was harvested 48 h postinoculation and checked for presence of virus by a hemagglutination assay. The virus was then plaque purified, and titers were determined on MDCK cells in the presence of 1.5 gofl-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin/ml. X-31 (H3N2) virus, A/Philippines/2/1982 (H3N2, Phil82) virus, A/Rhea/NC/39482/1993 (H7N1, RheaH7) virus, and ch5/3n1 (H5 HA globular head domain from A/Vietnam/1203/2004 virus and H3 HA stalk domain from A/Perth/16/2009 virus [Perth09]) virus (19) were grown in 10-day-old embryonated eggs, and titers were determined on MDCK cells. Sf9 insect cells (ATCC CRL-1711) were grown in TNM-FH medium (Gemini Bioproducts) supplemented with 10% fetal bovine serum (FBS), 1% Pluronic F68 (Sigma), and Pen/Strep (Gibco). BTI-TN-5B1-4 (High Five) insect cells (ATCC CRL-10859) were cultured in HyClone SFX serum-free insect cell medium (Fisher Scientific) containing Pen/Strep (Gibco). ch7/3n3 and ch5/3n1 both contained a regular human H3 cleavage site (located in the stalk domain) and were rescued in the A/Puerto Rico/8/34 backbone, which is considered safe for humans. All viruses were handled in biosafety level 2 facilities. Recombinant proteins. The ectodomains of ch7/3 HA (as described above), ch5/3 HA (H5 HA globular head domain from A/Vietnam/1203/ 2004 virus, H3 HA stalk domain from Perth09), ch4/3 HA (H4 HA head from A/duck/Czech/1956 virus, H3 HA stalk from Perth09), A/duck/ Czech/1956 H4 HA, A/Wisconsin/67/2005 (Wisc05) H3 HA, A/chicken/ Jalisco/12283/2012 H7 HA, A/Hong Kong/1/1968 H3 HA, Phil82 H3 HA, and Perth09 H3 HA were cloned in frame into a modified pfastbac baculo transfer vector (Invitrogen) that features a C-terminal T4 trimerization domain and a hexahistidine tag as described previously (21, 22). Briefly, baculovirus was generated using the FastBac system (Invitrogen) according to the manufacturer s recommendations. Recombinant baculovirus was amplified in Sf9 cells to P3 stocks, which were then used to infect High Five cells at a multiplicity of infection of 10. At 96 h postinfection, supernatants were harvested by low-speed centrifugation (2,000 rpm, 10 min, 4 C) and were incubated for 3 h with Ni-nitrilotriacetic acid (NTA) resin (Qiagen). The supernatant/slurry mixture was passed over gravity flow columns (Qiagen), washed with washing buffer (50 mm Na 2 HCO 3, 300 mm NaCl, 20 mm imidazole, ph 8), and eluted in 1-ml fractions (elution buffer included 50 mm Na 2 HCO 3, 300 mm NaCl, and 250 mm imidazole, ph 8). The eluted protein was then concentrated and buffer exchanged against phosphate-buffered saline (PBS; ph 7.4) using Amicon Ultra centrifugal filter units (Millipore) with a 30-kDa molecular mass cutoff in a swinging bucket rotor. Protein purity and integrity were assessed by SDS- PAGE and Western blot, and the protein concentration was measured using Bradford reagent (Bio-Rad). Experimental infection of mice and passive transfer experiments. All animal experiments were done in full compliance with the guidelines of the Mount Sinai School of Medicine Institutional Animal Care and Use Committee (IACUC). Animals were housed in facilities with a 12-hour dark and light cycle and free access to water and food. Groups of five sixto eight-week-old female BALB/c mice were anesthetized by intraperitoneal injection of 0.1 ml of ketamine/xylazine mixture (0.15 mg/kg and 0.03 mg/kg) and were intranasally inoculated with 10 4 plaque-forming units (PFU) of X-31, Phil82, or RheaH7 virus. Control groups of mice were vaccinated intramuscularly with inactivated Phil82 (1 g), a commercially available split vaccine that had A/Victoria/210/2009 (A/Perth/ 16/2009-like strain) as the H3 component (Fluzone ), or were DNA vaccinated using a TriGrid in vivo electroporation device (Ichor Medical Systems) with 80 g of pcaggs plasmid coding for Wisc05 (23). Subsets of X-31-infected mice were infected with 10 6 or 10 5 PFU of Phil82 4 weeks after the initial infection. Four weeks postinfection, mice were anesthetized and exsanguinated by terminal bleeding (intracardiac puncture). For passive transfer experiments, groups of five six- to eight-week-old female BALB/c mice were intraperitoneally injected with 250 l of serum per mouse from either naive mice or mice that were sequentially infected with X-31 (10 4 PFU) followed by Phil82 (10 5 PFU). Two hours posttransfer, mice were anesthetized by intraperitoneal injection of 0.1 ml of ketamine/xylazine mixture (0.15 mg/kg and 0.03 mg/kg) and were intranasally inoculated with 10 50% murine lethal doses (mld 50 ) of ch5/3n1 virus. Weight was monitored for 11 days, and mice that lost more than 25% of their initial body weight were scored dead. ELISA. Ninety-six-well enzyme-linked immunosorbent assay (ELISA) plates (Immulon 4 HBX) were coated with 0.1 g per well of purified recombinant HA protein in bicarbonate/carbonate coating buffer (100 mm, ph 9.6). Plates were then blocked for 1 h with PBS containing 0.1% Tween 20 (vol/vol) (TPBS) with 3% milk powder for the mouse serum ELISA or with TPBS containing 0.5% milk powder and 3% goat serum for the human serum assays. Serum was prediluted 1:100 (mouse) or 1:50 (human), serially diluted 1:3 (mouse) or 1:2 (human) in blocking buffer, and then adsorbed onto plates for 1 or 3 h, respectively. After extensive washing with TPBS, the bound antibody was detected with alkaline phosphatase (AP)-linked anti-mouse or anti-human IgG antibodies (both from Invitrogen) diluted 1:5,000 for 1 h at room temperature (RT), followed by another extensive washing step and detection with p- nitrophenyl phosphate (PNPP) substrate (Sigma). The reactions were stopped with a 0.75 M NaOH solution and read at 405 nm on a Synergy 4 (BioTek) plate reader. Human sera. Deidentified human sera from H3N2-infected individuals or from people who received the trivalent vaccine (Fluzone; H3 component A/Victoria/210/2009 [A/Perth/16/2009-like strain]) were collected in accordance with the internal review boards (IRB) of the University of Rochester (principal investigator [PI] John Treanor, IRB approval number RSRB 21870) and the Baylor Institute for Immunology Research (PI Karolina Palucka, IRB approval numbers and ), respectively. For each patient from the H3N2-infected cohort (n 7) (data not shown), we obtained a set of three samples collected as fol- April 2013 Volume 87 Number 8 jvi.asm.org 4729

3 Margine et al. lows: (i) one sample was collected in the fall of 2010, prior to influenza season; (ii) if patients experienced influenza-like symptoms, they were asked to return, infection was confirmed by PCR analysis, and a sample was subsequently collected 28 days after infection; (iii) the last sample was collected in May to June 2010, after the end of the influenza season. Two sets of samples from individuals with confirmed influenza B virus infections were used as controls. For the vaccinated cohort, all subjects were administered the Fluzone vaccine and samples were collected on day 0 before vaccination. Subjects returned to have postvaccination blood drawn 26 to 29 days later. All subjects provided informed consent. VNA. Virus neutralization assays (VNAs) were performed as described elsewhere (24). Briefly, 2-fold dilutions of receptor-destroying enzyme (Sigma)-treated sera (in sterile Opti-MEM [Invitrogen]) were mixed with 200 PFU of Perth09. The serum-virus samples were then incubated at RT for 60 min to allow any HA-specific antibodies present in the serum to neutralize the influenza virus. The serum-virus samples were then transferred onto MDCK monolayers cultured in 96-well flat-bottom plates. Following virus absorption for 60 min, the serum-virus inocula were removed, and the MDCK cells were cultured for 4 days in Opti-MEM supplemented with 1 g/ml of TPCK-trypsin (Sigma). Virus production was determined by HA assay. The neutralization titer is defined as the reciprocal of the highest dilution of serum that neutralizes 200 PFU of influenza virus. Immunoglobulin G purification. Mouse or human serum was diluted 1:10 in PBS and was filtered through a m filter unit. Immunoglobulin G (IgG) was then purified as described previously using a 4 Fast Flow Sepharose G resin (GE Healthcare) on a gravity flow column (Qiagen) (25). Briefly, diluted and filtered serum was passed over the column three times, the resin was then washed with four column volumes of PBS, and IgG was eluted using a 0.1 M glycine/hcl buffer at ph 2.7. The eluate was immediately neutralized with 2 M Tris-HCl buffer at ph 10. Purified IgG was then concentrated and buffer exchanged against PBS using Amicon Ultra centrifugal filter units (Millipore) with a 30-kDa molecular mass cutoff in a swinging bucket rotor. The protein concentration was measured with a NanoDrop 2000 spectrophotometer using the A 280 method. Plaque reduction neutralization assay. Chimeric H7/3N3 virus (100 PFU) was incubated in MEM medium with serial dilutions of purified human or mouse IgG (starting dilution, 600 g/ml; 5 5-fold dilution steps) for 1 h at RT in volumes of 100 l. The virus-igg mixture was then transferred onto MDCK cells in 12-well plates and incubated for 45 min. After the incubation period, the virus and IgG mixture was removed and the cells were overlaid with an agar-mem mixture containing matched concentrations of IgG, as well as TPCK-treated trypsin and DEAE dextrane and antibiotics (Pen/Strep). An anti-h7 antiserum (BEI NR and BEI NR-9226, 1:2 mixture; starting concentration, 1:100) was used as a positive control. Wells that were incubated with virus without IgG served as a negative control. Assays were incubated for 48 h at 37 C, fixed with 0.5% formalin, and stained using anti-h7 goat serum (BEI NR and BEI NR-9226), a horseradish peroxidase (HRP)-conjugated anti-goat antibody (Southern Biotech), and TrueBlue HRP substrate (KPL). Statistical tests and data analysis. All data plots and statistical analyses were generated using Prism4 (GraphPad). All values are plotted as averages with standard deviations of the means. Correlations were analyzed by fitting a linear regression line to the collected data and calculating the correlation coefficient (R 2 ) values. P values at or below 0.05 are considered statistically significant. For endpoint titers, the cutoff was considered to be the mean optical density (OD) measured for secondary antibody-only wells plus three standard deviations. RESULTS Infection with H3N2 viruses induces an HA stalk-specific antibody response in mice. We previously showed that infection with group 1 HA-expressing influenza viruses can induce HA stalkdirected antibodies (20) and that the titers can be greatly boosted upon a secondary infection with a different virus strain. This boost is most efficient when the HAs expressed by the two viruses have substantially different head domains but express conserved stalk regions (20, 21, 26, 27). We wanted to investigate how these processes translate to members of the group 2 influenza viruses, which generally share a high overall amino acid similarity and for which only a couple of epitopes have been described within the stalk domain (16 18). For this purpose, we infected BALB/c mice sublethally with two different H3N2 viruses Phil82 and X-31 or with RheaH7 (10 4 PFU per infection). Four weeks postinfection, animals were bled and sera were tested for levels of stalk-directed antibodies by an ELISA that included as the substrate a soluble chimeric HA construct with the head domain of an H4 virus and the stalk domain of an H3 strain (ch4/3) (19). All virus infections induced robust anti-stalk antibody responses; X-31 infection induced marginally higher titers than did Phil82 or RheaH7 infection (Fig. 1A). The higher antibody response following X-31 infection is likely attributable to higher replication levels of this high-yielding, reassortant vaccine strain in the mouse model (20, 28). We next wanted to determine whether a second infection can further boost the stalk-directed response elicited by the primary infection. For this purpose, separate groups of mice infected with 10 4 PFU of X-31 were infected 4 weeks later with 10 6 or 10 5 PFU of the Phil82 seasonal H3N2 influenza virus strain. Higher doses were chosen for the Phil82 infection to avoid clearance of the virus by the memory immune response elicited by the X-31 infection. The two viruses share an overall 91% amino acid identity, with the most conserved regions being in the stalk domain. We thus expected that the memory B cells recognizing epitopes in this region would be activated by the secondary Phil82 infection. The second exposure with the high dose triggered an almost-30-fold boost in the level of stalk antibodies compared to the titers elicited by the initial infection alone (Fig. 1A). This effect was dose dependent since the lower dose (10 4 ) only slightly increased the anti-stalk titer. In contrast, sera from animals vaccinated with a seasonal commercial trivalent influenza virus vaccine (TIV), vaccinated with an inactivated Phil82 virus, or electroporated with plasmid DNA encoding an H3 HA protein (Wisc05) had minimal to no reactivity to the stalk domain of the HA (Fig. 1B), although all of them seroconverted to homologous antigen to similar degrees (Fig. 2). As further assessment of the breadth of this humoral response, we analyzed the binding of sera from the different animal groups to HA proteins from H4 and H7 group 2 influenza virus subtypes. Cross-reactivity to the H4 HA was detected for sera from X-31- infected mice, with a 10-fold boost in antibody titers elicited upon the secondary infection with the Phil82 drift variant (Fig. 1C). No cross-reactivity for the H7 antigen was detected following the primary infection; however, a strong boost in broadly reactive antibodies was detected upon the secondary one. The primary antibody response may have been below the limit of detection due to a small number of epitopes shared between H7 and the other HAs. However, the secondary exposure probably resulted in reactivation and expansion of the B cells recognizing these epitopes, which subsequently led to the great boost in cross-reactive antibody titers we detected. As expected, the anti-h7 levels elicited by sequential infections with the seasonal H3 viruses were still lower than the antibody response obtained by infection with the homol jvi.asm.org Journal of Virology

4 Stalk-Reactive Antibodies Induced by H3N2 Infections Downloaded from FIG 1 Infection with influenza virus induces a stalk-specific humoral response in mice. (A) Mice (n 5) were sublethally infected with 10 4 PFU of Phil82 (H3 Phil ), X-31 (H3 X ), or RheaH7 (H7 Rhea 10 4 ) virus. Additional groups of X-31-infected mice were infected 4 weeks later with 10 6 or 10 5 PFU of the Phil82 drifted strain (H3 X H3 Phil or H3 X H3 Phil , respectively). Sera were collected from all animals 4 weeks after the last infection. Stalk-reactive antibodies were detected by measuring binding of sera to ch4/3 HA protein (Perth09 H3 stalk domain and H4 head domain) in an ELISA. Serum from naive mice was used as a negative control (naive sera). (B) ELISA stalk reactivity (ch4/3 substrate) of mice that were vaccinated intramuscularly (i.m.) with either an inactivated Phil82 virus preparation (inact. virus Phil82; 1 g total protein) or seasonal TIV vaccine (TIV vaccine; 1 g of the H3 component) or with DNA encoding Wisc05 HA (DNA Wisconsin/05 H3 HA; 80 g, electroporation). Values from X-31-infected animals from panel A are shown as positive controls. (C) Reactivity of the elicited antibodies to H4 hemagglutinin (from A/duck/Czech/1956). (D) Reactivity of the elicited antibodies to H7 hemagglutinin (from A/chicken/Jalisco/12283/2012). (E) Plaque reduction neutralization assay with ch7/3n3 virus and purified IgG from sera shown in panel A. The virus (100 PFU) was preincubated with 5-fold dilutions of purified IgG preparations (starting concentration, 600 g/ml) for 1hatRTprior to infection of a monolayer of MDCK cells. Agar overlay contained the respective dilutions of IgG. Polyclonal sera raised against an H7N7 virus were used as positive controls. (F) Passive transfer experiment with sera from sequentially infected mice (H3 X H3 Phil ) or naive mice (naive). Animals were challenged 2 h after transfer with a ch5/3n1 virus, and weight was monitored for 11 days. Numbers on the side of the legend indicate numbers of surviving mice out of the numbers of mice per group. ogous RheaH7 virus (Fig. 1D). Again, sera from animals that were vaccinated with inactivated virus, commercial TIV, or DNA encoding the Wisc05 HA showed no cross-reactivity toward either H4 or H7 HA protein (Fig. 1C and D), indicating that productive virus replication is required for stimulating these cross-reactive antibody responses in this model. Stalk-directed antibodies have neutralizing activity in vitro and in vivo. To determine the functionality of these stalk-reactive antibodies, we tested their neutralizing activity in a plaque reduction assay with an influenza virus expressing a chimeric HA with an H3 stalk and an H7 globular head (ch7/3), together with an N3 NA. Since mice were exposed only to H3N2 viruses, we reasoned that any in vitro inhibition of the ch7/3n3 virus would be the result of neutralizing antibodies directed against the stalk domain of the HA (H3). We purified total IgG antibodies from sera of mice that either experienced a single infection with the X-31 virus or were also exposed to a secondary infection with the Phil82 virus 4 weeks later. At high IgG concentrations (above 124 g/ml), sera from both H3 infected groups completely inhibited virus replication (Fig. 1E). However, at lower concentrations, we detected a significant difference in neutralizing activities of the two IgG preparations. Even at concentrations as low at 1 g/ml, purified IgG preparations obtained from animals that were exposed to two sequential H3N2 infections (X-31/Phil82) neutralized more than 70% of the ch7/3n3 virus, compared to only 20% being inhibited by the same amount of IgG purified from the X-31 prime-only animals (Fig. 1E). These differences mirror the results of the ELISA binding assays (Fig. 1B) and indicate that the induced stalkdirected antibodies are functional and can successfully neutralize virus infectivity. on November 10, 2018 by guest April 2013 Volume 87 Number 8 jvi.asm.org 4731

5 Margine et al. FIG 2 Reactivity of mouse sera to homologous full-length HAs. Sera from groups of mice shown in Fig. 1 were analyzed for reactivity to homologous HA protein. Mice (n 5) were sublethally infected with 10 4 PFU of Phil82 (H3 Phil ) and X-31 (H3 X ) viruses. Additional groups of X-31-infected mice were infected 4 weeks later with 10 6 or 10 5 PFU of the Phil82 drifted strain (H3 X H3 Phil or H3 X H3 Phil , respectively). Groups of mice were also vaccinated with either an inactivated Phil82 virus preparation (IV H3 Phil82; 1 g total protein) or seasonal TIV vaccine (TIV vaccine; 1 g of the H3 component) or with DNA encoding Wisc05 HA (DNA Wisconsin/05 H3 HA; 80 g, electroporation). Serum from naive mice was used as a negative control (naive sera). (A) ELISA reactivity of X-31-infected animals to homologous HA (from A/Hong Kong/1/1968). (B) ELISA reactivity of Phil82-infected or -vaccinated animals to homologous HA (from A/Philippines/2/1982). (C) ELISA reactivity of TIV-vaccinated animals to homologous HA (from A/Perth/16/ 2009). (D) ELISA reactivity of Wisc05 DNA-vaccinated animals to homologous HA (from A/Wisconsin/67/2005). Sera from sequentially infected animals (10 4 PFU X-31 followed by 10 5 PFU Phil82) or from naive mice were transferred into new sets of naive mice, and these mice were then challenged with 10 mld 50 of ch5/3n1 virus. Similarly to the ch7/3n3 virus used in the plaque reduction assay, the ch5/3n1 virus allows for measurement of anti-stalk antibodies without interference from antibodies directed to the H3 head domain or the N2 NA. The ch5/3n1 virus was chosen since, in contrast to ch7/3n3 virus, it is considerably pathogenic in the mouse model. Mice that received serum from sequentially infected animals lost minimal amounts of weight and were 100% protected from mortality, whereas animals in the control group lost weight quickly and succumbed to infection by day eight postinfection (Fig. 1F). Collectively, the data suggest that infection with group 2 influenza viruses elicits neutralizing stalk-directed antibody responses, which can be further boosted by a second infection with a drifted viral strain. Infection with a seasonal H3N2 virus induces stalk-directed antibodies in humans. The results obtained in the mouse model encouraged us to go one step further and investigate whether a similar induction of stalk antibodies occurs upon infection with seasonal H3N2 viruses in humans as well. For this purpose, we obtained matched samples from patients with confirmed influenza virus infections. Three serum samples were available for each individual (n 7): one collected before the influenza season, one obtained 28 days after onset of a confirmed influenza virus infection, and a third one following the influenza season that year. Two sets of samples from influenza B virus-infected individuals were included as negative controls. To confirm infection with an H3N2-type virus in these patients, we first tested the sera for an increase in reactivity to H3 HA proteins of Perth09 (Fig. 3A and B) and Wisc05 (data not shown) vaccine strains, which share very high amino acid identity. Seroconversion to an H3 HA was detected for all 9 patients within the observation period, confirming that these individuals had been infected with a seasonal H3N2 virus. To measure stalk-directed antibodies in their serum, we made use of chimeric HA constructs expressing an H3 stalk domain (from Perth09) and an H5 or H7 globular head domain (ch5/3 or ch7/3, respectively). Since humans are not generally exposed to H5 or H7 virus, we hypothesized that these individuals should have no preexisting antibodies directed against these globular heads and that the present constructs would allow us to specifically measure stalk reactivity. ELISA analysis of patient sera with these constructs showed that each individual had variable baseline titers of stalk antibodies; however, during the influenza season, these stalk-reactive antibody levels were boosted in all patients with confirmed H3N2 infection (Fig. 3C and D) (ch7/3 reactivity not shown). As a measure of the longevity of these responses, we did not detect a significant decrease in stalk antibody titers between samples collected immediately postillness and those collected after the end of the season in any of the patients. Since the data from the mouse experiments suggested that the degree of stalk antibody induction depends at least to some extent on the level of viral replication, we were curious to see whether there is a correlation between the general seroconversion to full-length HA glycoprotein (Fig. 3A and B) and stalk reactivity (Fig. 3C and D). Interestingly, when performing a linear regression and correlation analysis of the two data sets, we found a strong correlation (R , P ) between the levels of 4732 jvi.asm.org Journal of Virology

6 Stalk-Reactive Antibodies Induced by H3N2 Infections antibodies reactive to the stalk domain of the HA and the level of seroconversion, as measured by reactivity to the full HA protein (Fig. 3E) (R , P ). As a negative control, we measured ELISA reactivity to an irrelevant protein and correlated it with the reactivity to full-length H3 HA. Expectedly, we did not find a correlation between these data (R , P ), confirming the significance of our conclusion. Taken together, these analyses strengthen the results obtained from the mouse experiments and support the hypothesis that viral replication mediates induction of an antibody response directed to the stalk domain of the HA protein. In addition, we obtained samples collected from 11 people who received the TIV in the 2009 to 2010 season. For each individual, we obtained matched serum samples collected before and 4 weeks after vaccine administration. By verifying neutralization titers in April 2013 Volume 87 Number 8 each sample against the H3N2 vaccine strain, we confirmed that all individuals but one had responded to the vaccine, albeit to various degrees (data not shown). We then measured the levels of stalk antibodies in the serum samples by ELISA using a ch5/3 HA construct. Samples from two randomly chosen patients (FAM047 and FAM153) from the infected cohort were taken along as positive controls for the assay, while samples from a noninfected individual (FAM157) were used as negative controls. While the induction of stalk-reactive antibody titers detected in the infected patients (Fig. 4) was reproducibly similar to that shown in Fig. 3, we did not see a boost of stalk-directed antibodies in the sera of any of the TIV vaccine recipients. These data are consistent with the earlier results obtained from analysis of sera from mice upon administration of a similar vaccine (Fig. 1B) and, together with the analysis of the sera from the infected patients, support our hy- jvi.asm.org 4733 FIG 3 Infection with seasonal H3N2 virus induces stalk-directed antibodies in humans. (A and B) Sera from patients who presented with influenza-like illness in the 2010 to 2011 season were analyzed for seroconversion by ELISA with recombinant Perth09 HA as the substrate. For each patient, reactivity of samples collected before the influenza season, immediately after illness, or after the season was analyzed. The two negative-control data sets are grouped on the left side of the panel (influenza B infected). Data shown are OD values from samples diluted 1:200 (A) or endpoint titers from the same assay (B). (C and D) The same set of samples was analyzed for HA stalk reactivity by using a chimeric HA construct expressing a group 2 stalk (Perth09) and an H5 head (ch5/3). The two negative-control data sets are grouped on the left side of the panel (influenza B infected). Data shown are OD values from samples diluted 1:200 (C) or endpoint titers from the same assay (D). (E) The induction of stalk-directed antibodies correlates with the level of seroconversion as measured by reactivity to the full-length HA protein (R , P ).

7 Margine et al. FIG 4 Vaccination with seasonal inactivated trivalent vaccines does not induce an anti-ha stalk humoral response in humans. (A) Serum samples obtained from patients who received the inactivated trivalent vaccine in the 2009 to 2010 season were analyzed by ELISA for stalk reactivity using recombinant ch5/3 protein as the substrate. For each patient, matched samples obtained before vaccination and 4 weeks after were analyzed in the same assay. Data shown are from samples diluted 1:200. (B) Data in panel A are represented as fold inductions by plotting the ratio obtained by dividing the signal in the postvaccination sample by the signal obtained for the sample collected before vaccination. pothesis that replicating virus infection is a more efficient way to trigger a stalk-specific response. Stalk-directed antibodies elicited in humans have neutralizing activity. In order to assess the functionality of the stalk-directed antibodies induced upon H3N2 infection in humans, we measured their ability to block viral infectivity in vitro. To allow for a quantitative comparison between samples, and also to remove sialylated molecules, we used purified total IgG for neutralization studies. The ch7/3n3 influenza virus was used for the plaque reduction assays to ensure that only neutralizing activity directed against the stalk domain of the HA protein was measured. The five highest responders (FAM153, FAM292, FAM047, FAM193, and FAM046) according to the ELISA data (Fig. 3) were examined, and serum from an uninfected patient was used as a negative control (FAM157). For each patient, purified IgG preparations were obtained from the preseason and postillness samples. As shown in Fig. 5, and consistent with the ELISA data, serum collected in the preseason period had variable neutralizing activity in each patient. The levels of neutralizing activity, however, were significantly increased upon infection in all patients that were H3N2 seropositive (Fig. 3A and B). The only exception was patient FAM292, whose preinfection serum already had very high neutralization activity. This was in agreement with the ELISA data, which also indicated very high levels of stalk-directed antibodies in the preseason sample for this patient (Fig. 3C and D). In addition, it is of note that the ELISA reactivity to the stalk domain correlates strongly with the neutralizing activity of purified IgG preparations (Fig. 5B). Taken together, these data suggest that the antibody response directed against the stalk of group 2 HAs, which is moderately boosted by seasonal influenza virus infection, has neutralizing activity and is able to inhibit viral replication. DISCUSSION Here we show that primary infection with group 2 HA-expressing influenza viruses X-31 (H3), Phil82 (H3), and RheaH7 (H7) induces robust stalk antibody responses in mice. This effect was not observed in naive mice vaccinated with H3 HA-encoding DNA or inactivated virus preparations, including the commercially available seasonal trivalent vaccine. This difference may be explained by potentially higher levels of antigen presentation as a result of increased HA surface expression by infected cells during productive infection, better exposure of the stalk domain in the native conformation, and/or increased presentation of translation products during infection (29). Alternatively, infection of dendritic cells may further improve activation of B cells through cross-presentation and stimulation of helper T cells. A similar antigen presentation profile should theoretically be achieved by DNA vaccination as well, with plasmid-transfected cells expressing HA trimers in the native conformation on their surface. However, attempts to induce detectable levels of stalk antibodies in mice electroporated with DNA encoding an H3 HA protein were unsuccessful, a result which is similar to previous attempts to induce group 1 stalk antibodies (20). Our results therefore suggest that live attenuated vaccines and viral vectors may be more efficient in induction of stalk-reactive antibodies than inactivated vaccines. Recent reports of novel H3N8 strains circulating among harbor seals (30), human infections by swine origin H3N2 variant strains, and sporadic human infections with H4, H7, and H10 avian influenza viruses have raised concerns about future influenza pandemics (31 35). These concerns may be mitigated by the development of novel influenza vaccines that provide universal coverage against multiple influenza subtypes. In an attempt to assess the level of cross-reactive epitopes within the stalk domains of group 2 HA-expressing influenza virus subtypes, we tested the ability of the antibodies elicited by H3N2 virus infection to bind to purified H4 and H7 subtype HA proteins. Interestingly, the stalkdirected response elicited by the primary H3N2 infections exhibited a low degree of reactivity against the representative heterosubtypic HAs. A second infection with an antigenically drifted H3N2 virus, however, induced a significant boost of the crossreactive response. This indicates that group 2 HAs share epitopes within the stalk domain that can be successfully targeted by a humoral immune response. The binding pattern of the detected polyclonal antibodies resembles, in principle, the binding profile of the broadly neutralizing human monoclonal antibody CR8020, which binds to a membrane-proximal epitope on the group 2 stalk (17). The induced stalk-specific antibodies may efficiently neutralize virus infection and may be recalled from memory B cells upon a second antigen encounter. Furthermore, when used in a passive transfer experiment, they were able to protect mice from morbidity and mortality, demonstrating neutralizing activity in vivo. In order to verify the relevance of the results obtained from the mouse experimental model, human serum samples collected from individuals who either experienced an H3N2 virus infection or received the inactivated seasonal trivalent vaccine in the 2009 to 4734 jvi.asm.org Journal of Virology

8 Stalk-Reactive Antibodies Induced by H3N2 Infections FIG 5 The anti-stalk response boosted by seasonal H3N2 infection in humans has neutralizing activity. The five highest responders were chosen for this analysis (as determined by ELISA). For each individual, total IgG was purified from the sample collected before onset of the influenza season as well as from the postillness sample. To measure stalk-directed neutralizing activity, we used a virus that expresses an HA with an H3 stalk and an H7 head as well as an N3 NA (ch7/3n3). The virus (100 PFU) was incubated with IgG dilutions for 1 h at RT before infection of monolayers of MDCK cells. For all samples, the starting IgG concentration used in the assay was 600 g/ml, and 5 5-fold dilutions were considered. The respective IgG concentrations were maintained in the agar overlay. Serum from an uninfected individual (Fam157) was used as an uninfected negative control. Polyclonal sera raised against an H7N7 virus were used as positive controls, and PBS was used as a negative control. (B) Correlation between ELISA reactivity and neutralizing activity. OD values from the 1:200 dilution in ELISA using ch5/3 protein were plotted against neutralizing activity at a 125- g/ml IgG dilution, and a correlation analysis was performed (R , P 0.001). April 2013 Volume 87 Number 8 jvi.asm.org 4735

9 Margine et al season were analyzed for stalk-reactive antibodies. As determined from the antibody responses of H3N2 virus-infected mice, infection, but not vaccination with seasonal inactivated preparations, boosted cross-reactive stalk antibody responses in humans as well. This boost in titers of cross-reactive antibodies, however, was more modest than the boost detected in humans upon infection with pandemic H1N1 viruses, where neutralizing titers against the stalk were boosted up to 30-fold (21). The differences in stalk-reactive antibody responses following pandemic H1N1 infection versus seasonal H3N2 infection can be explained by the introduction of an antigenically distinct H1N1 virus in the human population, which efficiently replicated in the absence of preexisting antibodies. Also, the high amino acid identity of the head domains of the H3N2 drift variants preserves shared antigenic epitopes in the globular head domain. Susceptibility to H3N2 infection may thus have been reduced in an antigen-experienced population and may have stimulated the selective expansion of head-specific antibodies over stalk-reactive antibodies. We assume that higher levels of replication lead to more antigen expression in the host and, therefore, to a higher level of seroconversion, which would fit well with the observation that induction of stalkreactive antibodies in infected humans correlates well with the degree of reactivity to full-length H3 proteins. Importantly, we were also able to show that this stalk-directed response was sufficient to neutralize viral infection in vitro and in vivo. These observations are relevant for the development of universal influenza virus vaccines and are encouraging, as they suggest that broadly neutralizing stalk-reactive antibodies against group 2 HAs can be boosted in humans. ACKNOWLEDGMENTS We thank Chen Wang for excellent technical support. Florian Krammer was supported by an Erwin Schrödinger fellowship (J 3232) from the Austrian Science Fund (FWF). This work was supported by CEIRS (Centers for Excellence for Influenza Research and Surveillance; HHSN C [Mount Sinai School of Medicine] and HHSN C [University of Rochester Medical Center]) as well as by HIPC (Human Immunology Project Consortium; grant number 1U19AI ). REFERENCES 1. Simonsen L, Reichert TA, Viboud C, Blackwelder WC, Taylor RJ, Miller MA Impact of influenza vaccination on seasonal mortality in the US elderly population. Arch. Intern. Med. 165: Centers for Disease Control and Prevention Update: influenza activity United States, season and composition of the influenza vaccine. MMWR Morb. Mortal. Wkly. Rep. 61: Centers for Disease Control and Prevention Update: influenza activity United States, season, and composition of the influenza vaccine. MMWR Morb. Mortal. Wkly. 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