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. Author manuscript; available in PMC: 2013 Oct 10.
Published in final edited form as: Virology. 2012 Jun 22;432(1):39–44. doi: 10.1016/j.virol.2012.06.003

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

Judith D Easterbrook a,#, Louis M Schwartzman a, Jin Gao b, John C Kash a, David M Morens c, Laura Couzens b, Hongquan Wan b, Maryna C Eichelberger b, Jeffery K Taubenberger a
PMCID: PMC3725556  NIHMSID: NIHMS384774  PMID: 22727831

Summary

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.

Introduction

There has been concern that a highly pathogenic avian influenza (HPAI) virus of the recent Eurasian H5N1 lineage, which has been associated with a case-fatality rate of about 59% (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2011_08_09/en/index.html), could adapt to humans and become pandemic (Peiris, de Jong, and Guan, 2007). Fortunately, little human-to-human transmission has been observed thus far (Nguyen, Farrar, and Horby, 2008), and serosurveillance studies in high-risk human populations (e.g. contact with poultry in enzootic regions, sick birds, or patients infected with H5N1) report low prevalences of H5N1 antibodies (Beigel et al., 2005; Wang, Parides, and Palese, 2012).

Vaccines against H5N1 have made significant progress, but challenges remain, including poor immunogenicity, low cross-clade reactivity, and issues regarding biosafety containment and high pathogenicity when grown in eggs (Horimoto and Kawaoka, 2009; Kang et al., 2009). Virus-like particles (VLP) are a promising technology because they are non-infectious and present structurally native and highly antigenic protein conformations that induce robust antibody, as well as increased CD8+ and CD4+ T cell, responses (Bright et al., 2007; Kang et al., 2009; Quan et al., 2007). Preliminary data from clinical trials with a H5N1 influenza VLP vaccine further indicate immunogenicity and non-reactogenicity (Khurana et al., 2011).

Influenza NA cleaves sialic acids from cell surface glycans to release progeny virus and prevent post-release viral aggregation, resulting in reduced lung titers and pathology (Air, 2011; Johansson, Bucher, and Kilbourne, 1989; Kilbourne et al., 2004). Currently, the potency of influenza vaccines is measured by single radial immunodiffusion (SRID) that quantifies only HA. NA content is not quantified and NA immunogenicity is usually not measured. Commercially derived inactivated influenza vaccines that have elicited good anti-HA antibody responses may induce little or no anti-NA antibody responses (Aymard et al., 2003; Easterbrook et al., 2010). NA is thus potentially attractive for inclusion in vaccines because NA immunity contributes to limiting infection and is strongly associated with reduction in viral replication and reduced disease severity. Further, antigenic drift is slower than HA drift (Kilbourne, Johansson, and Grajower, 1990; Sandbulte et al., 2011) and, once elicited, NA antibodies decline at lower rates than HA antibodies (Johansson, Bucher, and Kilbourne, 1989).

The importance of NA in influenza immunity was suggested epidemiologically in 1968 when it was shown that individuals immune to previously circulating H2N2 viruses were protected against infection with the new H3N2 pandemic virus by anti-N2 antibodies that had been elicited by H2N2 viruses circulating between 1957 and 1968 (Monto and Kendal, 1973; Murphy, Kasel, and Chanock, 1972). Several studies have evaluated cross-protection between influenza viruses of the same NA, but different HA, subtype, which cannot fully discount effects of heterosubtypic immunity afforded by HA or robust T cell responses to conserved epitopes (Bright et al., 2008; Ichinohe et al., 2007; Perrone et al., 2009). Further, due to the dominance of HA on the virus surface, HA immunogenicity outcompetes NA in the context of co-expression (Air, 2011; Johansson et al., 1987; Kendal, Noble, and Dowdle, 1977) and this study included evaluation of protection afforded by NA in the absence of HA competition.

Influenza VLPs consisting of NA and a small amount of M form particles that are similar in size and morphology to influenza virions, with oligomeric NA present on the VLP surface (Lai et al., 2010; Saito, Taylor, and Webster, 1995). In this study, we evaluated cross-reactivity of antibodies elicited by the NA of 2009 pandemic H1N1 (H1N1pdm09) with the NA of H5N1, and assessed the ability of NA-expressing VLPs to protect mice against H5N1 influenza infection. This is the first study specifically using NA VLPs as a vaccine, as well as the first to assess the protection afforded by the NA of the H1N1pdm09 against H5N1 infection.

Results and Discussion

Characterization of the NA VLPs

The coding sequence of the NA gene of the H1N1pdm09 (CA/09) and H5N1 (VN/1203) viruses share 82% nucleotide identity and 87.7% identity at the amino acid level. The NA VLPs were spheroidal and approximately 50–200 µm in diameter, similar in morphology to influenza virions (Figure 1) and similar to a previous published full characterization of VLPs expressing only NA and M (Lai et al., 2010). When standardized by the amount of protein, the activity of CA/09 NA VLPs was about 2-fold higher than VN/1203 NA VLPs for both the small NA-XTD substrate (1704 and 879 mU/mg protein, respectively), as well as for the larger fetuin substrate (576 and 341 mU/mg protein, respectively), consistent with previously reported higher NA enzymatic activities of H1N1pdm09 viruses as compared with highly pathogenic H5N1 viruses (Lin et al., 2009; Wu et al., 2010). NA proteins are inherently unstable, and may be even less stable when originating from H5N1 viruses because several potential glycosylation sites and a cysteine that are likely to contribute to tetrameric stability are deleted in the short-stalk NA of HPAI H5N1 viruses (Air, 2011; Blok and Air, 1982). To better define the relationship between NA expression on the virus surface, NA activity and immunogenicity, however, requires further investigation.

Figure 1. TEM of VLPs expressing NA and M proteins.

Figure 1

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VLPs comprised of the NA from VN/1203 (A), CA/09 (B), and M genes of NY312 were negatively stained and viewed by a transmission electron microscope at 242,000× magnification.

CA/09 VLP-vaccinated mice were protected from a lethal challenge with VN/1203 and had reduced lung titers and pathology

In this study, protection afforded by immunization with NA-containing VLPs was evaluated in a lethal highly pathogenic H5N1 influenza virus challenge model in mice. Vaccination with the homologous VN/1203 NA VLP completely protected mice from an otherwise lethal infection with the homologous virus and these mice also lost no weight after challenge (Figure 2). Mice vaccinated with the CA/09 NA VLP lost only a moderate amount of weight (maximum 12%) during infection and all survived (Figure 2). This experiment was repeated to verify the results and taken out to 18 days post-challenge, by which time all mice vaccinated with CA/09 NA VLPs fully recovered any body weight that had been lost (maximum 5.4% weight loss; data not shown). Mice that were previously infected with CA/09 all survived challenge with VN/1203 and lost no weight (Figure 2). Mock-vaccinated mice did not survive a challenge with VN/1203 and lost weight, succumbing to infection by 8 dpc (Figure 2).

Figure 2. Mice that were vaccinated with the VN/1203 or CA/09 NA VLPs or previously infected with CA/09 were completely protected from a lethal challenge with VN/1203.

Figure 2

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Mice (n=5/treatment group) were infected with CA/09 or were vaccinated and boosted with CA/09 or VN/1203 NA VLPs administered i.n. at 130 mU NA per vaccine dose. Six weeks later, mice were challenged with VN/1203 (10X LD50) and weight loss (A) and survival (B) were assessed for 14 days. Mice vaccinated with VN/1203 or CA/09 NA VLPs had greater survival and reduced weight loss as compared to mock-vaccinated mice, p < 0.05.

Mice that received the VN/1203 or CA/09 NA VLPs had approximately 100-fold and 10-fold less virus in the lungs than mock-vaccinated mice 4 dpc, respectively (Figure 3; p<0.05). Mice that were previously infected with CA/09 did not have any detectable infectious virus in the lungs 4 dpc with VN/1203 (Figure 3; p<0.05). While the VLPs contained influenza matrix proteins, VLP-vaccinated animals did not elicit a detectable M2 antibody response (see methods).

Figure 3. Virus titers were reduced in mice vaccinated with the VN/1203 or CA/09 NA VLPs or absent in mice previously infected with CA/09.

Figure 3

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Infectious virus titers were evaluated in the lungs of mice 4 days post-challenge with 10X LD50 VN/1203 (n = 3/group). Mice had been vaccinated and boosted with VN/1203 or CA/09 NA VLPs administered at 130 mU NA, i.n. Only 1 of 3 mice vaccinated with VN/1203 NA VLP, and none of the mice previously infected with CA/09 had a measurable lung virus titer, as indicated by the numbers above the bars. *Vaccinated < mock-vaccinated, p < 0.05 and the dotted line indicates the limit of detection.

Mock-vaccinated mice displayed diffuse, acute, necrotizing bronchitis and bronchiolitis, with an acute, widespread alveolitis and prominent pulmonary edema on day 4 post-challenge (Figure 4A). Influenza viral antigen was widespread throughout the respiratory tree (Figure 4B) in bronchial, bronchiolar, and alveolar epithelium, and in alveolar macrophages. Less severe histopathologic changes were observed in mice immunized with CA/09 NA VLPs, but in these mice, the lung sections showed acute bronchitis and bronchiolitis, though only focal alveolitis and viral antigen was focally associated with lesions (Figure 4C). In mice previously infected with CA/09, only focal, mild acute bronchiolitis and focal acute inflammation of alveolar ducts was observed, coupled with a low prevalence of viral antigen. Mice vaccinated with VN/1203 NA VLPs showed only focal pathologic changes with focal, mild acute bronchitis and bronchiolitis associated with rare, focal viral antigen (Figure 4D). Submucosal and perivascular lymphoid aggregates, indicative of antibody responses, were prominent in all mice that survived challenge with VN/1203, including CA/09 NA and VN/1203 VLP vaccinated (Figure 4C & 4D) and CA/09 infected mice. These data highlight the ability of immunogenic NA vaccines to mitigate severe influenza disease using intranasally-administered NA-expressing VLPs.

Figure 4. Pulmonary pathology caused by infection with H5N1 was reduced in mice vaccinated with VN/1203 or CA/09 NA VLPs.

Figure 4

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Photomicrographs of hematoxylin- and eosin-stained tissue sections and immunohistochemically stained sections to detect influenza viral antigen from mice mock-vaccinated or NA VLP-vaccinated and subsequently challenged with the HPAI VN/1203 virus at day 4 post-challenge. Viral antigen is stained red-brown on a hematoxylin-stained background (original magnifications 40×.) (A) Representative lung section from a mock-vaccinated mouse challenged with VN/1203 showing diffuse, acute, necrotizing bronchitis [B] and bronchiolitis [Br], with an acute, widespread alveolitis and prominent pulmonary edema. (B) Consecutive lung section from (A) showing abundant viral antigen in the bronchitis [B], bronchiolitis [Br], and alveolitis lesions [arrows]. (C) Representative lung section from a CA/09 NA VLP-vaccinated mouse showing reduced pathology and only focal viral antigen associated with mild bronchitis [B] and focal alveolitis [arrow]. (D) Representative lung section from an VN/1203 NA VLP-vaccinated mouse showing limited, focal pathology and only focal viral antigen associated with mild bronchitis [B] and focal alveolitis [arrows].

VLP vaccination elicited homologous humoral responses with detectable cross-reactivity to VN/1203 NA

NI titers were measured to evaluate functional antibodies elicited by NA VLP vaccinationor prior infection. Mice that were previously infected with CA/09 had the highest NI titers against the homologous NA (Table 1), and NI titers also were elicited against the homologous NA by vaccination with the NA VLPs (Table 1). Low levels of cross-reactive heterologous NI titers with the NA of VN/1203 were detectable by NA inhibition assay in 50% (5/10) of mice vaccinated with the CA/09 NA VLP and 80% (8/10) of mice that were previously infected with CA/09 (Table 1). A recent study similarly demonstrated cross-reactive NI titers to the HPAI H5N1, Hong Kong/213/2003, in ferrets vaccinated with a live-attenuated H1N1pdm09 influenza vaccine (GMT = 102) (Chen et al., 2012).

Table 1.

NI titers against VN/1203 after vaccination with VN/1203 or CA/09 NA VLPs or prior infection with CA/09

NI Titers (GMT)
Vaccine VN/1203 NA CA/09 NA
VN/1203 NA VLP 532 ± 138 -
CA/09 NA VLP 7.5 ± 0.8a 866 ± 150
Prior CA/09 Infection 31 ± 16 4664 ± 256
Mock <10 <10
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Mice were vaccinated and boosted with VN/1203 or CA/09 NA VLPs administered at 130 mU NA activity i.n., were mock-vaccinated (PBS), or were infected with CA/09. Serum was collected 5 weeks after the initial VLP vaccination or infection with CA/09. NI titers were measured against the appropriate homologous NA or the heterologous VN/1203 NA.

a

Only 50% in this group had a low titer, the remaining mice had a titer <10. For purpose of calculating GMT, titers <10 were assigned a value of 5.

Although detectable homologous NI titers were elicited in all mice, survival and the amount of infectious virus and pathology in the lungs did not correlate with homologous or cross-reactive NI titers against the NA of VN/1203. Similarly, vaccination with DNA encoding the NA of a seasonal H1N1, A/New Caledonia/99, protected 50% of mice against a lethal VN/1203 challenge, also in the absence of high cross-reactive IgG titers (detectable in only 4/32 mice) (Sandbulte et al., 2007). In contrast, however, we found no protection or reduction of virus titer or pathology in the lungs in mice vaccinated with VLPs expressing the NA of a 2009 seasonal H1N1 (A/Bethesda/NIH50/2009 (H1N1) [NIH50]) following challenge with VN/1203; however, these mice had detectable, though lower homologous NI titers than CA/09 and VN/1203 NA VLP vaccinated mice (38.7 ± 8 GMT; data not shown). This may be attributable to differences in dose, form of antigen, site of delivery, or to the higher degree of identity between the NAs of H5N1 and A/New Caledonia/99 (80.4% identity of amino acid) than the NAs of H5N1 and NIH50 (78.5% identity of amino acids). Protection correlated with amino acid identity, suggesting that this may be a predictor. We also cannot rule out the possibility that a higher dose of NIH50 NA VLPs might also induce partial cross-protection with the H5N1 NA.

Future studies will examine the contribution of NA antibodies in mediating cross-protection through passive immunization studies. While low levels of NA antibodies may contribute to heterologous protection, antibodies to conserved regions also may play a role through antibody-dependent cell-mediated cytotoxicity or cytokine secretion. Other factors, such as mucosal IgG and IgA antibodies or presence of activated CD8+ T cells at the effector site, could also contribute to protection after intranasal administration (Bessa et al., 2008; McBurney, Young, and Ross, 2007; Quan et al., 2007), and will be evaluated in future NA cross-protection experiments. Further, whether NA VLP are a commercially viable vaccine production system requires further investigation.

Materials and Methods

VLPs

The NA gene segments of A/California/04/2009 (H1N1) [CA/09] and A/Vietnam/1203/2004 (H5N1) [VN/1203] were each cloned into a pCAGGS expression plasmid and transfected into 293T cells along with the A/New York/312/2001 (H1N1) M gene segment in pCAGGS using Polyjet DNA transfection reagent (SignaGen, Rockville, MD). Cell culture supernatant was harvested after 72 h and debris was removed by centrifugation at 2,000 × g for 10 min. VLPs were concentrated by ultracentrifugation at 100,000 × g for 2 h and purified using a 20–60% discontinuous sucrose gradient at 130,000 × g for 16 h. The fractions that were positive for NA activity were collected and concentrated by ultracentrifugation at 100,000 × g for 2 h. Total protein was quantified using the Bradford BCA assay (Pierce, Rockford, IL) and the proportion of NA of the total protein was measured by Coomassie blue staining and semiquantitative densitometry analysis. The amounts of M and other cellular proteins incorporated into the VLP were determined to be approximately <25%; by western blot, no M2 was detectable. To verify that M2 antibodies were not elicited by vaccination, we previously measured IgG antibodies against ferrets and mice vaccinated with NA VLPs made with the same M, as described previously (Tompkins et al., 2007), and no M2 antibodies were detectable in vaccinated mice.

NA activity of the VLPs was measured using the NA-Star kit (Applied Biosystems, Foster City, CA) as a small substrate or the miniaturized assay to measure conversion of fetuin, as a larger substrate (Sandbulte et al., 2009), and was standardized to the known activity per units of the purified NA of Clostridium perfringens (Sigma, St Louis, MO). The structure and shape of the NA VLPs were confirmed by negative staining transmission electron microscopy (TEM).

Viruses

The fully reconstructed VN/1203 and CA/09 influenza viruses were prepared using a standard reverse genetics-based system (Tumpey et al., 2005). VN/1203 virus and infectious samples were handled under biosafety level 3 enhanced laboratory (BSL3+/ABSL3+) conditions in accordance with the Select Agent guidelines of the National Institutes of Health (NIH), the Centers for Disease Control and Prevention (CDC), and the United States Department of Agriculture (USDA).

Mouse Experiments

Groups of 7–8 week old female BALB/c mice (Jackson Laboratories, Bar Harbor, ME; n=10 per treatment group) were lightly anesthetized with isofluorane supplemented with O2 (1.5 L/min) before immunization or virus challenge. Mice (n = 10 per group) were immunized intranasally (i.n.) with 130 mU NA of the appropriate NA VLP (corresponding to 15.7 µg CA/09 NA VLP protein and 34.7 µg VN/1203 NA VLP protein) in 50 µl, in an attempt to elicit mucosal, as well as serum, antibody responses (Bessa et al., 2008). VLPs were standardized by activity because NA tetrameric stability correlates with activity and immunogenicity, not total protein, and standardized methods to quantify NA protein concentrations are lacking (Brett and Johansson, 2006; Kendal, Noble, and Dowdle, 1977). Three weeks after the initial vaccination, mice were boosted with the same amount of vaccine via the same route. One group of mice was also inoculated once, i.n., with a sublethal dose of CA/09 (104 pfu) under light anesthesia. Six weeks after the initial vaccination (3 weeks after boost) or infection, mice were anesthetized as described previously and challenged i.n. with 10x LD50 (50 pfu) of VN/1203 in 50 µl DMEM. Survival and body weight were monitored for 14 days and mice were humanely euthanized if more than 25% of initial body weight was lost. To verify the survival and weight loss results, this experiment was repeated for the group that received the CA/09 NA VLPs (n=5). Lungs were collected for viral titration (n=3 per vaccine group) and pathologic examination (n=2 per vaccine group) at 4 days post-challenge (dpc). Virus titers were measured by plaque assay using 10% (w/v) lung homogenates in sterile L15 media. All experimental work was performed in an enhanced ABSL3 laboratory at the NIH, following approval of animal safety protocols by the NIH Animal Care and Use Committee and in accordance with the Select Agent guidelines of the NIH, the CDC, and the USDA.

Neuraminidase Inhibition (NI) Assay

Serum was collected from mice 5 weeks after the first VLP vaccination or infection with CA/09. To measure NA inhibiting antibody titers, reassortant viruses containing the appropriate N1, an avian H6 HA, and the remaining segments from A/Puerto Rico/8/34 (H1N1) (PR8) were created using plasmid-based reverse genetics (Sandbulte et al., 2009). The NI assay was performed as described previously using fetuin as a NA substrate (Cate et al., 2010). The NA inhibition titer was defined as the inverse of the greatest dilution that gave at least 50% inhibition of NA activity.

Histopathological and Immunohistochemical Analyses

Following 24 h fixation in 10% formaldehyde, inflated lung samples were embedded in paraffin, cut into 5 µm sections, and mounted on positively charged slides (HistoServ, Germantown, MD). Influenza virus antigen distribution was evaluated by immunohistochemistry using a polyclonal goat anti-H1N1 (Abcam, Cambridge, MA), as described previously (Memoli et al., 2009). A single pathologist reviewed the histopathology and immunohistochemistry in a blinded fashion.

Statistical Analyses

Survival and mean time to death were analyzed by Kaplan-Meier survival analysis (Graph Pad Prism, La Jolla, CA). Differences in the maximum percent weight loss, lung virus titers, and antibody responses were assessed using the student’s t-test. Mean differences were considered statistically significant if p < 0.05.

Conclusions

Our data suggest that prior infection with H1N1pdm09 or vaccination with VLPs containing this NA protect against a lethal H5N1 infection. Antibodies to a homosubtypic NA might reduce disease severity and transmission, potentially blunting the impact of a pandemic if widely used in the population. In future influenza vaccine strategies, consideration should be given to inclusion of immunogenic forms of NA, which may provide expanded immunoprotection against endemic or pandemic influenza viruses.

Highlights.

  • NA VLPs induced protective immunity against challenge with a homologous H5N1 virus

  • VLPs containing the 2009 pandemic H1N1 NA protected mice against a lethal heterologous H5N1 challenge

  • Heterologous protection was afforded even in the absence of high levels of cross-reactive NI titers

Acknowledgements

We thank the Comparative Medicine Branch (NIH/NIAID) for their assistance with animal studies. We would also like to thank Dr. David Dorward at the Rocky Mountain Laboratories, NIAID, NIH for the TEM images and Aline Sandouk for the IHC staining. This work was supported in part by the Intramural Research Program of the NIH and the NIAID, by the BARDA MCM Initiative IV: Influenza Vaccine Manufacturing Improvement (JKT), and by FDA CBER PanFlu funds (MCE). We want to further acknowledge Dr. Edwin Kilbourne’s remarkable and important contributions to our understanding of neuraminidase immunity.

Footnotes

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References

  1. Air GM. Influenza neuraminidase. Influenza Other Respi Viruses. 2011 Nov 16; doi: 10.1111/j.1750-2659.2011.00304.x. Epub ahead of print. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aymard M, Ferraris O, Gerentes L, Jolly J, Kessler N. Neuraminidase assays. Dev Biol (Basel) 2003;115:75–83. [PubMed] [Google Scholar]
  3. Beigel JH, Farrar J, Han AM, Hayden FG, Hyer R, de Jong MD, Lochindarat S, Nguyen TK, Nguyen TH, Tran TH, Nicoll A, Touch S, Yuen KY. Avian influenza A (H5N1) infection in humans. N Engl J Med. 2005;353(13):1374–1385. doi: 10.1056/NEJMra052211. [DOI] [PubMed] [Google Scholar]
  4. Bessa J, Schmitz N, Hinton HJ, Schwarz K, Jegerlehner A, Bachmann MF. Efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design. Eur J Immunol. 2008;38(1):114–126. doi: 10.1002/eji.200636959. [DOI] [PubMed] [Google Scholar]
  5. Blok J, Air GM. Variation in the membrane-insertion and "stalk" sequences in eight subtypes of influenza type A virus neuraminidase. Biochemistry. 1982;21(17):4001–4007. doi: 10.1021/bi00260a015. [DOI] [PubMed] [Google Scholar]
  6. Brett IC, Johansson BE. Variation in the divalent cation requirements of influenza A virus N1 neuraminidases. J Biochem. 2006;139(3):439–447. doi: 10.1093/jb/mvj051. [DOI] [PubMed] [Google Scholar]
  7. Bright RA, Carter DM, Crevar CJ, Toapanta FR, Steckbeck JD, Cole KS, Kumar NM, Pushko P, Smith G, Tumpey TM, Ross TM. Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle. PLoS One. 2008;3(1):e1501. doi: 10.1371/journal.pone.0001501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bright RA, Carter DM, Daniluk S, Toapanta FR, Ahmad A, Gavrilov V, Massare M, Pushko P, Mytle N, Rowe T, Smith G, Ross TM. Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin. Vaccine. 2007;25(19):3871–3878. doi: 10.1016/j.vaccine.2007.01.106. [DOI] [PubMed] [Google Scholar]
  9. Cate TR, Rayford Y, Nino D, Winokur P, Brady R, Belshe R, Chen W, Atmar RL, Couch RB. A high dosage influenza vaccine induced significantly more neuraminidase antibody than standard vaccine among elderly subjects. Vaccine. 2010;28(9):2076–2079. doi: 10.1016/j.vaccine.2009.12.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen Z, Kim L, Subbarao K, Jin H. The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets. Vaccine. 2012;30(15):2516–2522. doi: 10.1016/j.vaccine.2012.01.090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Easterbrook JD, Kash JC, Sheng ZM, Qi L, Gao J, Kilbourne ED, Eichelberger MC, Taubenberger JK. Immunization with 1976 swine H1N1- or 2009 pandemic H1N1-inactivated vaccines protects mice from a lethal 1918 influenza infection. Influenza Other Respi Viruses. 2010;5(3):198–205. doi: 10.1111/j.1750-2659.2010.00191.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Horimoto T, Kawaoka Y. Designing vaccines for pandemic influenza. Curr Top Microbiol Immunol. 2009;333:165–176. doi: 10.1007/978-3-540-92165-3_8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ichinohe T, Tamura S, Kawaguchi A, Ninomiya A, Imai M, Itamura S, Odagiri T, Tashiro M, Takahashi H, Sawa H, Mitchell WM, Strayer DR, Carter WA, Chiba J, Kurata T, Sata T, Hasegawa H. Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine. J Infect Dis. 2007;196(9):1313–1320. doi: 10.1086/521304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johansson BE, Bucher DJ, Kilbourne ED. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virol. 1989;63(3):1239–1246. doi: 10.1128/jvi.63.3.1239-1246.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johansson BE, Moran TM, Bona CA, Kilbourne ED. Immunologic response to influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. III. Reduced generation of neuraminidase-specific helper T cells in hemagglutinin-primed mice. J Immunol. 1987;139(6):2015–2019. [PubMed] [Google Scholar]
  16. Kang SM, Pushko P, Bright RA, Smith G, Compans RW. Influenza virus-like particles as pandemic vaccines. Curr Top Microbiol Immunol. 2009;333:269–289. doi: 10.1007/978-3-540-92165-3_14. [DOI] [PubMed] [Google Scholar]
  17. Kendal AP, Noble GR, Dowdle WR. Neuraminidase content of influenza vaccines and neuraminidase antibody responses after vaccination of immunologically primed and unprimed populations. J Infect Dis. 1977;136(Suppl):S415–S424. doi: 10.1093/infdis/136.supplement_3.s415. [DOI] [PubMed] [Google Scholar]
  18. Khurana S, Wu J, Verma N, Verma S, Raghunandan R, Manischewitz J, King LR, Kpamegan E, Pincus S, Smith G, Glenn G, Golding H. H5N1 virus-like particle vaccine elicits cross-reactive neutralizing antibodies in humans that preferentially bind to oligomeric form of influenza hemagglutinin. J Virol. 2011;85(21):10945–10954. doi: 10.1128/JVI.05406-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kilbourne ED, Johansson BE, Grajower B. Independent and disparate evolution in nature of influenza A virus hemagglutinin and neuraminidase glycoproteins. Proc Natl Acad Sci U S A. 1990;87(2):786–790. doi: 10.1073/pnas.87.2.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kilbourne ED, Pokorny BA, Johansson B, Brett I, Milev Y, Matthews JT. Protection of mice with recombinant influenza virus neuraminidase. J Infect Dis. 2004;189(3):459–661. doi: 10.1086/381123. [DOI] [PubMed] [Google Scholar]
  21. Lai JC, Chan WW, Kien F, Nicholls JM, Peiris JS, Garcia JM. Formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase. J Gen Virol. 2010;91(Pt 9):2322–2330. doi: 10.1099/vir.0.019935-0. [DOI] [PubMed] [Google Scholar]
  22. Lin X, Zhou J, Zhang Y, Wu J, Zhang F, Li Z, Bi S, Shu Y, Wang Y. Oseltamivir boosts 2009 H1N1 virus infectivity in vitro. Biochem Biophys Res Commun. 2009;390(4):1305–1308. doi: 10.1016/j.bbrc.2009.10.142. [DOI] [PubMed] [Google Scholar]
  23. McBurney SP, Young KR, Ross TM. Membrane embedded HIV-1 envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes. Virology. 2007;358(2):334–346. doi: 10.1016/j.virol.2006.08.032. [DOI] [PubMed] [Google Scholar]
  24. Memoli MJ, Tumpey TM, Jagger BW, Dugan VG, Sheng ZM, Qi L, Kash JC, Taubenberger JK. An early 'classical' swine H1N1 influenza virus shows similar pathogenicity to the 1918 pandemic virus in ferrets and mice. Virology. 2009;393(2):338–345. doi: 10.1016/j.virol.2009.08.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Monto AS, Kendal AP. Effect of neuraminidase antibody on Hong Kong influenza. Lancet. 1973;1(7804):623–625. doi: 10.1016/s0140-6736(73)92196-x. [DOI] [PubMed] [Google Scholar]
  26. Murphy BR, Kasel JA, Chanock RM. Association of serum anti-neuraminidase antibody with resistance to influenza in man. N Engl J Med. 1972;286(25):1329–1332. doi: 10.1056/NEJM197206222862502. [DOI] [PubMed] [Google Scholar]
  27. Nguyen TH, Farrar J, Horby P. Person-to-person transmission of influenza A (H5N1) Lancet. 2008;371(9622):1392–1394. doi: 10.1016/S0140-6736(08)60494-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Peiris JS, de Jong MD, Guan Y. Avian influenza virus (H5N1): a threat to human health. Clin Microbiol Rev. 2007;20(2):243–267. doi: 10.1128/CMR.00037-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Perrone LA, Ahmad A, Veguilla V, Lu X, Smith G, Katz JM, Pushko P, Tumpey TM. Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge. J Virol. 2009;83(11):5726–5734. doi: 10.1128/JVI.00207-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Quan FS, Huang C, Compans RW, Kang SM. Virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus. J Virol. 2007;81(7):3514–3524. doi: 10.1128/JVI.02052-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Saito T, Taylor G, Webster RG. Steps in maturation of influenza A virus neuraminidase. J Virol. 1995;69(8):5011–5017. doi: 10.1128/jvi.69.8.5011-5017.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sandbulte MR, Gao J, Straight TM, Eichelberger MC. A miniaturized assay for influenza neuraminidase-inhibiting antibodies utilizing reverse genetics-derived antigens. Influenza Other Respi Viruses. 2009;3(5):233–240. doi: 10.1111/j.1750-2659.2009.00094.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sandbulte MR, Jimenez GS, Boon AC, Smith LR, Treanor JJ, Webby RJ. Cross-reactive neuraminidase antibodies afford partial protection against H5N1 in mice and are present in unexposed humans. PLoS Med. 2007;4(2):e59. doi: 10.1371/journal.pmed.0040059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sandbulte MR, Westgeest KB, Gao J, Xu X, Klimov AI, Russell CA, Burke DF, Smith DJ, Fouchier RA, Eichelberger MC. Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses. Proc Natl Acad Sci U S A. 2011;108(51):20748–20753. doi: 10.1073/pnas.1113801108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tompkins SM, Zhao ZS, Lo CY, Misplon JA, Liu T, Ye Z, Hogan RJ, Wu Z, Benton KA, Tumpey TM, Epstein SL. Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg Infect Dis. 2007;13(3):426–435. doi: 10.3201/eid1303.061125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tumpey TM, Basler CF, Aguilar PV, Zeng H, Solorzano A, Swayne DE, Cox NJ, Katz JM, Taubenberger JK, Palese P, Garcia-Sastre A. Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science. 2005;310(5745):77–80. doi: 10.1126/science.1119392. [DOI] [PubMed] [Google Scholar]
  37. Wang TT, Parides MK, Palese P. Seroevidence for H5N1 influenza infections in humans: Meta-analysis. Science. 2012;335(6075):1463. doi: 10.1126/science.1218888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wu J, Zhang F, Wang M, Xu C, Song J, Zhou J, Lin X, Zhang Y, Wu X, Tan W, Lu J, Zhao H, Gao J, Zhao P, Wang Y. Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses. PLoS One. 2010;5(12):e15825. doi: 10.1371/journal.pone.0015825. [DOI] [PMC free article] [PubMed] [Google Scholar]

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