# Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses.

Judit Szécsi, Bertrand Boson, Per Johnsson, Pia Dupeyrot-Lacas, Mikhail Matrosovich, Hans-Dieter Klenk, David Klatzmann, Viktor Volchkov, Cosset François-Loïc

<figure><img src="image_1.png" /></figure>

## To cite this version:

Judit Szécsi, Bertrand Boson, Per Johnsson, Pia Dupeyrot-Lacas, Mikhail Matrosovich, et al. Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses. Virol J, 2006, 3, pp.70. 10.1186/1743-422X-3-70. inserm-00097847

HAL Id: inserm-00097847

https://inserm.hal.science/inserm-00097847

Submitted on 22 Sep 2006

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
Short report

Open Access

# Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses

Judit Szécsi¹,²,³, Bertrand Boson¹,²,³, Per Johnsson¹,²,³, Pia Dupeyrot-Lacas¹,²,³,⁴, Mikhail Matrosovich⁵, Hans-Dieter Klenk⁵, David Klatzmann⁶, Viktor Volchkov¹,²,³ and François-Loïc Cosset*¹,²,³

Address: ¹INSERM, U1758, F-69007 Lyon, France, ²Ecole Normale Supérieure de Lyon, F-69007 Lyon, France, ³IFR128 BioSciences Lyon-Gerland, F-69007 Lyon, France, ⁴Epixis SA, Lyon, F-69007 Lyon, France, ⁵Institut fur Virologie, Universitätsklinikum Giessen und Marburg, D-35033 Marburg, Germany and ⁶Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, CNRS-UMR7087, Université Pierre et Marie Curie, Hôpital Pitié-Salpêtrière, 83 Bd de l'Hôpital, 75013 Paris, France

Email: Judit Szécsi - judit.szecsi@ens-lyon.fr; Bertrand Boson - bboson@ens-lyon.fr; Per Johnsson - per.johnsson@ens-lyon.fr; Pia Dupeyrot-Lacas - Pia.Dupeyrot-Lacas@ens-lyon.fr; Mikhail Matrosovich - Mikhail.Matrosovich@med.uni-marburg.de; Hans-Dieter Klenk - Klenk@med.uni-marburg.de; David Klatzmann - david.klatzmann@chups.jussieu.fr; Viktor Volchkov - volchkov@cervi-lyon.inserm.fr; François-Loïc Cosset* - flcosset@ens-lyon.fr

Published: 03 September 2006

Virology Journal 2006, 3:70 doi:10.1186/1743-422X-3-70

This article is available from: http://www.virologyj.com/content/3/1/70

© 2006 Szécsi et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 16 August 2006

Accepted: 03 September 2006

## Summary

There is an urgent need to develop novel approaches to vaccination against the emerging, highly pathogenic avian influenza viruses. Here, we engineered influenza viral-like particles (Flu-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two highly pathogenic influenza viruses of either H7N1 or H5N1 antigenic subtype. We demonstrate that, upon recovery of viral RNAs from a field strain, one can easily generate expression vectors that encode the HA, NA and M2 surface proteins of either virus and prepare high-titre Flu-VLPs. We characterise these Flu-VLPs incorporating the HA, NA and M2 proteins and we show that they induce high-titre neutralising antibodies in mice.

Influenza virus infects thousands of people each year, causing epidemics with severe mortality [1]. Moreover, there is an increasing concern about a potential influenza pandemic, as highly virulent avian influenza strains are spreading from South-East Asia, with a high risk to cross species-specific barriers [2]. With such a menace, we should be well prepared to prevent excessive mortality, should a virulent pandemic occur.

Vaccination, so far, has been the best manner to protect individuals from influenza infection. Influenza vaccines

have been used for ca. 50 years [3]. Current influenza vaccines are mostly inactivated formulations relying on the antigenic activity of the surface glycoproteins of influenza virus: a hemagglutinin (HA) and a neuraminidase (NA) [4]. A major problem, when preparing an influenza vaccine, is the lack of cross-immunity generated against different influenza virus subtypes. This is due to the high mutagenic capacity of influenza virus to generate forms that can escape the immune system. Antigenic shifts and antigenic drifts are evolutionary mechanisms that lead to serologically different influenza virus subtypes or strains

<footnote>* Corresponding author</footnote>
against which a vaccine is not efficient [5]. Thus, new vac-
cines need to be prepared during each seasonal influenza
epidemic and, importantly, there is no vaccine against the
novel, emerging highly pathogenic viruses. Influenza vac-
cines are generally produced from virus grown in embry-
onated chicken eggs. This implies that manufacturing a
vaccine preparation, from the appearance of a new sub-
type of influenza virus until the readiness of the vaccine,
takes several months [6]. Moreover, one needs to modify
the HA of highly virulent influenza strains in order to be
able to produce vaccines without killing the embryos.
Recently, reverse genetic methods have been used to pro-
duce vaccines in cell culture [6,7]. Finally, in the event of
a pandemic, the vaccine production has to be massive,
quick and safe.

Altogether, there is a strong need for developing novel
immunogenic formulations that can rapidly be prepared
as vaccines against the emerging highly pathogenic avian
influenza virus. As a step along this road, here we describe
a novel influenza virus immunogen using engineered
viral-like particles (Flu-VLPs) that mimic the properties of
the viral surface of two highly pathogenic influenza
viruses of H7N1 and H5N1 subtypes.

We used the surface proteins HA, NA and M2 of two
highly pathogenic avian influenza viruses: A/Chicken/
FPV/Rostock/1934 (H7N1) [8] and A/Thailand/KAN-1/
04 (H5N1) [9,10] to generate Flu-VLPs (H7-VLPs and H5-
VLPs, respectively). The influenza hemagglutinin is
responsible for virion attachment to the target cells
through recognition and binding to terminal sialic acid
groups on membrane-bound proteins of the host cell
(reviewed in [11]). The neuraminidase destroys non-func-
tional receptors to which hemagglutinin can bind and
thus facilitates virus access to target cells at the early stages
of infection and promotes egress of progeny viral particles
from infected cells late in infection [12,13]. M2 is a small
transmembrane protein with an ion channel activity
which regulates the pH inside the virion during viral entry
into cell and protects newly synthesized acid-labile H5
and H7 hemagglutinins during their transport through
low pH cellular compartments (reviewed in [14,15]).
Cloning of expression vectors for HA, NA and M2 from
H7N1 virus has been described elsewhere [8,16-19]. The
human virus isolate of H5N1, A/Thailand/KAN-1/04
(H5N1) [9], was kindly provided by Pilaipan Putha-
vathana at Mahidol University, Bangkok, Thailand. We
made one passage of the original seed virus in MDCK cells
and isolated viral RNA using the High Pure RNA isolation
kit (Roche Molecular Biochemicals, Mannheim, Ger-
many). HA, NA and M2 coding sequences were then
amplified from total viral RNA using Superscript Reverse
transcriptase and specific primers (sequences are available
upon request). PCR products were introduced into a CMV

promoter-driven expression plasmid in a manner identi-
cal to that used for H7N1 [18].

Flu-VLPs were assembled on replication-defective core
particles derived from murine leukaemia virus (MLV). For
immunisation purposes, they consisted of empty "core"
particles generated by the sole expression of MLV Gag pro-
teins [20], whereas for infectious assays, they comprised
MLV GagPol proteins and a recombinant genome encod-
ing the green fluorescent protein (GFP) [16,19]. Transduc-
tion of this marker gene in 'infected' target cells and
expression of GFP in transduced cells is indeed an accurate
reflection of the infection steps mediated by the surface
glycoproteins of retrovirus-derived VLPs [17,21,22] and,
hence, is a convenient way to study cell entry and neutral-
isation of highly pathogenic viruses in category 2 labora-
tories [23-25]. We produced Flu-VLPs harbouring at their
surface HA, HA and either NA or M2, or all three proteins
derived from the H7N1 or H5N1 viruses, by transient
expression in 293T cells of surface (HA, NA, M2) and
internal (Gag, GFP marker genome) viral components.
Expression of the different viral proteins in producer cells
and their incorporation on sucrose cushion-purified viral
particles was characterized by Western blot using specific
primary antibodies (Fig. 1A). All proteins were readily
expressed in producer cells (not shown). The hemaggluti-
nin, detected as uncleaved HA₀ precursor and HA₁/HA₂
cleaved mature forms, was incorporated on the surface of
the viral particles at high levels for both H7-VLPs and H5-
VLPs, when expressed together with NA (Fig. 1A). The
neuraminidase and M2 protein were also detected on
purified viral particles (Fig. 1A), yet at low levels as com-
pared to their expression in producer cells (data not
shown).

The expression of M2 during Flu-VLP production did not
influence the incorporation of HA or NA onto the viral
particles (Fig. 1A). In contrast, only small amounts of HA
proteins were detected on particles when NA was not co-
expressed in producer cells, correlating with low quanti-
ties of MLV Gag-derived capsid (CA) proteins (Fig. 1A).
This was most likely due to a less effective release of VLPs
into the cell supernatant in the absence of NA. Indeed
treatment of these latter cells with purified neuraminidase
from Vibrio cholerae induced efficient release of the viral
particles (data not shown). This confirmed the essential
role of NA to promote the release virus particles from the
cell surface by removing sialic acid receptors from pro-
ducer cells [12,26].

To estimate the concentration of Flu-VLPs, we determined
their 'transduction titres' by adding serial dilutions of viral
particle preparations harbouring a GFP marker gene to
TE671 human rhabdomyosarcoma cells. The medium
was then replaced with normal culture medium and the
A.

<figure><img src="image_3.png" /></figure>

B.

<figure><img src="image_4.png" /><figcaption>**Figure 1**</figcaption></figure>

**Biochemical and functional analysis of Flu-VLPs.** A. Incorporation of HA, NA and M2 proteins from H7N1 (A/Chicken/FPV/Rostock/1934) or H5N1 (A/Thailand/KAN-1/04) influenza viruses on MLV retroviral core particles, as indicated. Purified VLPs were loaded on SDS-PAGE and incorporation levels of the different proteins were determined by Western Blot analysis using polyclonal sera against H7N1-HA, H5N3-HA, H7N1-NA and M2 proteins. HA₀: HA precursor protein, HA₁: HA surface subunit; HA₂: HA transmembrane subunit; NA: neuraminidase; CA: MLV capsid protein. The difference in the molecular weight of H5 vs. H7 HA₂, of ca. 3 kDa, was due to the presence of an additional glycan for H7 HA₂. B. Infectious titres obtained with Flu-VLPs incorporating surface proteins from H7N1 or H5N1 influenza viruses or with VSV-VLPs incorporating the VSV-G of vesicular stomatitis virus (VSV), as indicated. VLP-containing supernatants were used to infect 10⁵ TE671 target cells plated in 12-well for 6 hr at 37°C. The transduction efficiency of GFP, determined as the percentage of GFP-positive cells, was measured by fluorescence-activated cell sorter (FACS) analysis 72 hr after infection, as previously described [22]. The transduction titres, provided as GFP-transducing units (t.u.)/ml, were calculated by using the formula: $Titre = %inf \times (10^5/100) \times d$, where "d" is the dilution factor of the viral supernatant and "%inf" is the percentage of GFP-positive cells as determined by FACS analysis using dilutions of the viral supernatant that transduced between 0.1% and 5% of GFP-positive cells. Due to the use of a FACS method to monitor transduced cells, the determination of GFP-positive cell below 0.1%, i.e., corresponding to transduction titres below 10³ t.u./ml in our experimental conditions, could not be accurately be determined. The background levels of transduction were therefore fixed at 10³ t.u./ml in all experiments.
transduction titre was deduced 72 hr later from the per-
centage of GFP-positive cells measured by fluorescence-
activated cell sorter (FACS) analysis, as previously
described [22].

In accordance with biochemical data (Fig. 1A), Flu-VLPs produced in the absence of NA displayed relatively low transduction titres, below 10⁵ t.u./ml (Fig. 1B). Consistent with its effect on HA incorporation (Fig. 1A), the presence of NA increased the infectivity by about 100 fold for H7-VLPs and 1,000 fold for H5-VLPs, raising transduction titres higher than those obtained with VLPs harbouring VSV-G (VSV-VLPs), one of the most efficient viral surface glycoprotein in such assays [17]. Of note, the infectivity of the Flu-VLPs was specifically and completely abolished by immune sera from animals inoculated with wild type influenza virus (Fig. 2A).

Interestingly, the transduction titres obtained with H7-
VLPs were about 50-fold lower than those obtained with
H5-VLPs (Fig. 1B). Furthermore, incorporation of M2
onto the Flu-VLPs increased the infectivity of H7-VLPs by
about 10 times (Fig. 1B), as reported previously [27], but
not that of H5-VLPs, as similar H5 HA incorporation lev-
els were reached irrespective of whether or not M2 was
expressed (Fig. 1A). This suggested that H7 HA, but not
H5 HA, was sensitive to M2 functions. Consistent with its
capacity to regulate the internal pH of endosomal com-
partments, the role of M2 during Flu-VLP production is
probably to prevent acidification and premature activa-
tion of HA protein, an event for which H7N1 virus HA is
apparently more sensitive than HA of H5N1 virus strain
used in this study.

Altogether, these results indicated that HA, NA and M2
incorporated onto the surface of VLPs are functional as
they efficiently mediate cell entry.

We then investigated whether Flu-VLPs harbouring all three viral proteins can induce specific immune responses and neutralising antibodies in mice. For these studies, H7-VLPs or H5-VLPs were concentrated and purified by ultra-centrifugation [19] before injection in BalbC mice. Control VLPs, incorporating the VSV-G glycoprotein [17,18], were also prepared and injected to mice in parallel. As an attempt to induce cross-neutralising antibodies against different influenza strains, we generated H7-VLPs or H5-VLPs treated with a citrate buffer at pH5.3 for 10 minutes. Indeed, at low pH, HA undergoes irreversible conformational changes that are required to induce membrane fusion [28]. Such conformational changes alter the structure and antigenicity of HA [29,30] and may result in exposure of conserved epitopes, hidden in the native HA conformation, that could induce cross-neutralising antibodies that are not raised otherwise, particularly in con-

served regions of HA₂ [31]. Conformational changes were
verified by demonstration of a complete loss of infectivity
by low pH-treated particles (data not shown) [28].

About 10⁸ particles of H7-VLPs or H5-VLPs, treated or non-treated at low pH, as well as VSV-VLPs particles were repeatedly injected intraperitoneally in 5 week-old female BalbC mice at 2 weeks intervals. The sera were harvested 2 weeks after each injections (harvests S1, S2, S3 and S4) and were decomplemented by heat inactivation at 56°C for 1 hr. We next determined the neutralising activity of the sera using the Flu-VLPs or the VSV-VLPs harbouring a GFP marker gene. The results of a typical experiment shown in Fig. 2A, are displayed as the % of neutralisation of the S2 sera compared to the S0 pre-immune sera, i.e., sera harvested before the first inoculation for each mouse, for a 1/100 dilution of these sera. Sera from mice injected with H7-VLPs neutralised specifically H7-VLPs, but neither the H5-VLPs nor the VSV-VLPs and *vice-versa*. Sera from mice injected with native Flu-VLPs neutralised more efficiently the homologous Flu-VLPs than sera from mice injected with acid pH-denatured Flu-VLPs; yet no cross-neutralisation was observed for the latter sera. Consistently, as tested on immunoblots of H7-VLPs vs. H5-VLPs, no cross-reactivity of H7- and H5-VLP sera could be observed for HA. Antibodies against M2 that detected M2 from either influenza virus strain were raised in some immunised mice (Fig. 2B), in agreement with the strong sequence homology between H7 M2 and H5 M2. No cross-reacting NA antibodies could be detected (Fig. 2B), perhaps owing to the relatively inefficient incorporation of this glycoprotein on the Flu-VLPs. Only few other non-specific protein bands were observed (Fig. 2B), suggesting that the antibody response against Flu-VLPs was specific. To investigate how the neutralising titres increased after repeated immunisations, we determined the titration curves for each serum harvest. The results are shown in Fig. 2C as the mean neutralisation values from sera of the different groups of mice. The neutralisation curves were similar for both H7 and H5 sera. We found that S1 sera, harvested 2 weeks after the first injection, had significant neutralising activity, with 50% neutralising activity-reached at the 1/500 serum dilution and with an ID₉₀ at the 1/100 dilution. The S2, S3 and S4 sera had much higher neutralising activities, even at high dilutions, with ID₉₅ obtained at the 1/2,500 dilution for the S3 and/or S4 sera.

Altogether these results indicated that retroviral-derived
VLPs incorporating HA, NA and M2 influenza proteins are
able to induce antibody production in mice. Moreover,
the immune response induced by these particles is rapid
and robust, achieving efficient neutralisation only two
weeks after the first injection. The produced antibodies are
specific, as no cross-reaction between different influenza
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2**</figcaption></figure>

**Immunogenicity of Flu-VLPs.** A. Neutralising activity of S2 sera from mice immunised with Flu-VLPs incorporating the HA, NA and M2 proteins from H7N1 (lanes 1–8) or H5N1 (lanes 13–20) influenza viruses (H7-VLP and H5-VLP, respectively), or with VSV-VLPs harbouring the VSV-G glycoprotein (lanes 9–12). Some Flu-VLPs were treated at acidic pH5.3 (H7-VLP (A) and H5-VLP (A)) to induce irreversible conformational changes in the HA protein before injection (lanes 5–8 and lanes 13–16 for H7-VLPs and H5-VLPs, respectively). Sera from each mouse were incubated at 37°C for 40 min with H7-VLPs, H5-VLPs or VSV-VLPs harbouring a GFP marker gene, as indicated, and then used to infect TE671 target cells. The transduction titres determined in the presence of diluted mouse sera were calculated as described in Fig. 1. The results are expressed as the mean percentages (mean ± SD; n = 5) of neutralisation of the transduction titres determined with the immune sera relative to titres determine with S0 pre-immune sera. Sera or antibodies raised against FPV (fowl plague virus; H7N1 influenza virus) and VSV were used as controls in the neutralisation assays (anti-FPV and anti-VSV, respectively). B. Determination of the specificity of antibodies raised in S2 sera from mice immunised with Flu-VLPs by Western blot analysis. H7-VLPs (left) and H5-VLPs (right) were loaded onto SDS-PAGE and transferred to membrane after electrophoresis. Lanes of these membranes were individualised and separately incubated with S2 sera from immunised mice (see above) at a 1/500 serum dilution. S0: pre-immune serum; S. HA; control polyclonal rabbit serum raised H7N1 HA; S. M2; control serum raised against H7 M2; HA₀: HA precursor protein; HA₁: HA surface subunit; HA₂: HA transmembrane subunit; NA: neuraminidase; M2: M2 matrix protein. C. The neutralising curves of sera from mice immunised with H7-VLPs and H5-VLPs, harvested two weeks after each injection (S1, S2, S3 and S4), were determined for different serum dilutions (1/20, 1/100, 1/500 and 1/2,500). The results are expressed as the mean percentages (mean ± SD; n = 5) of neutralisation of the transduction titres determined with the immune sera relative to titres determine with S0 pre-immune sera.
26. Bosch V, Kramer B, Pfeiffer T, Starck L, Steinhauer DA: Inhibition of release of lentivirus particles with incorporated human influenza virus haemagglutinin by binding to sialic acid-containing cellular receptors. *J Gen Virol* 2001, **82**:2485-2494.

27. McKay T, Patel M, Pickles RJ, Johnson LG, Olsen JC: Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors. *Gene Ther* 2006, **13**:715-724.

28. Skehel JJ, Bayley PM, Brown EB, Martin SR, Waterfield MD, White JM, Wilson IA, Wiley DC: Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. *Proc Natl Acad Sci U S A* 1982, **79**:968-972.

29. Graves PN, Schulman JL, Young JF, Palese P: Preparation of influenza virus subviral particles lacking the HA1 subunit of hemagglutinin: unmasking of cross-reactive HA2 determinants. *Virology* 1983, **126**:106-116.

30. Sagawa H, Ohshima A, Kato I, Okuno Y, Isegawa Y: The immunological activity of a deletion mutant of influenza virus haemagglutinin lacking the globular region. *J Gen Virol* 1996, **77** (Pt 7):1483-1487.

31. Okuno Y, Isegawa Y, Sasao F, Ueda S: A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. *J Virol* 1993, **67**:2552-2558.

32. Yao Q, Kuhlmann FM, Eller R, Compans RW, Chen C: Production and characterization of simian--human immunodeficiency virus-like particles. *AIDS Res Hum Retroviruses* 2000, **16**:227-236.

33. Duisit G, Salvetti A, Moullier P, Cosset FL: Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines. *Human Gene Therapy* 1999, **10**:189-200.

34. Roberts ML, Wells DJ, Graham IR, Fabb SA, Hill VJ, Duisit G, Yuasa K, Takeda S, Cosset FL, Dickson G: Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle. *Hum Mol Genet* 2003, **11**:1719-1730.

35. Savard N, Cosset FL, Epstein AL: Use of defective HSV-1 vectors harbouring gag, pol, and env genes to rescue defective retrovirus vectors. *J Virol* 1997, **71**:4111-4117.

Publish with **BioMed Central** and every
scientist can read your work free of charge

"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."

Sir Paul Nurse, Cancer Research UK

Your research papers will be:

* available free of charge to the entire biomedical community

• peer reviewed and published immediately upon acceptance

* cited in PubMed and archived on PubMed Central

* yours — you keep the copyright

Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp

<figure><img src="image_8.png" /></figure>

BioMedcentral