WO2008138120A1

WO2008138120A1 - Avian influenza h5n1 hemagglutilin pseudotyped lentiviral vector system for rapid identification of antivirals and neutralizing polypeptides

Info

Publication number: WO2008138120A1
Application number: PCT/CA2008/000886
Authority: WO
Inventors: Xiojian Yao; Zhujun Ao; Gary Kobinger; Darwin Kobasa
Original assignee: University Of Manitoba; Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health
Priority date: 2007-05-11
Filing date: 2008-05-12
Publication date: 2008-11-20

Abstract

Influenza glycoproteins HA and NA were efficiently incorporated onto HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Simultaneous expression of NA and M2 maximized vector production and transduction. Interestingly, the H5N1-pseudotyped HIV vector was sensitive to oseltamivir (tamiflu, an NA inhibitor), amantadine (an M2 inhibitor) or HA peptides independently. This H5N1 -pseudotyped HIV vector has been adapted for high- throughput screening of drug candidates such as small molecules, peptides or antibodies which can be safely performed in a biosafety level 2 environment. The H5N1 -pseudotyped HIV vector system mimics the influenza virus entry process and can greatly accelerate drug discovery to prepare against the next pandemic.

Description

Avian influenza H5N1 hemagglutilin pseudotyped lentiviral vector system for rapid identification of antivirals and neutralizing polypeptides PRIOR APPLICATION INFORMATION

The present application claims the benefit of US Provisional Patent Application 60/917,391 , filed May 11 , 2007. BACKGROUND OF THE INVENTION

The avian influenza H5N1virus is a subtype of the Influenza A virus that can cause illness in humans and many other animal species. The earliest infections of humans by H5N1 coincided with an epizootic of H5N1 influenza in Hong Kong's poultry population, infecting 18 people with a fatality rate of 33% (Subbarao, Klimov et al. 1998; Chan 2002). Since then, the H5N1 virus has spread to over 60 countries or regions and infected several million wild and domestic birds. Between 2003 and Feb 2007, there were a total of 272 confirmed human cases of H5N1 viral infection, 166 of which were fatal, according to the World Health Organization (World Health Organization 2007). At present, even though H5N1 viruses are not readily transmissible between humans, it is quite possible that they can acquire such transmissibility via mutations and/or gene reassortment from circulating human influenza A viruses. Due to the high virulence of this virus and its endemic presence as well as its significant ongoing mutations, the H5N1 virus is the world's largest current pandemic threat and preparations are being made for a potential epidemic.

Although vaccination is the primary means for controlling influenza pandemics, antivirals provide an additional line of defense, particularly important for controlling a rapidly spreading pandemic (Ferguson, Cummings et al. 2005; Longini, Nizam et al. 2005). Currently, two classes of influenza antivirals are available: inhibitors for viral M2 ion channel proteins (amantadine and rimantadine) and neuraminidase inhibitors (zanamavir and oseltamivir). However, their extensive use coupled with the recombination potential of the influenza genome has promoted the emergence of drug resistant strains (Bright, Shay et al. 2006), (Kiso, Mitamura et al. 2004). There are several in vitro assays available to analyze HA binding to human-type receptors and to identify compounds with specific antiviral properties such as receptor specificity assay by using a solid-phase binding assay with the sodium salts of sialylglycopolymers (Yamada, Suzuki et al. 2006). In addition M2 and NA activity and function can be evaluated with the silico screening channel function assay and in vitro assays for viral NA activity respectively (Fornabaio, Cozzini et al. 2003)(Giffin, Racier et al. 1995; Tai, Escarpe et al. 1998). These analyses are based on in vitro assays generating information that requires confirmation with cell-based functional assays which involve manipulation of live H5N1 virus in a BSL-3 laboratory. Recently, the influenza HA-pseudotyped lentiviral/MLV vectors have been successfully used to study gene transfer into lung epithelial cells or vaccination against H5N1 virus (Neumann, Watanabe et al. 2000; Kobinger, Weiner et al. 2001 ; McKay, Patel et al. 2006; Szecsi, Boson et al. 2006).

SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins in the presence of an agent of interest under conditions where in the absence of the agent the influenza HA, M2 and NA and the additional viral proteins would assemble into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles under conditions suitable for infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles. According to a second aspect of the invention, there is provided a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins under conditions suitable for assembly into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles in the presence of an agent of interest where in the absence of the agent the virus-like particles would infect the cells capable of infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles.

According to a second aspect of the invention, there is provided a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins in the presence of an agent of interest under conditions where in the absence of the agent the influenza HA₁ M2 and NA and the additional viral proteins would assemble into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles under conditions suitable for infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles.

According to a third aspect of the invention, there is provided a method of treating an H5N1 influenza infection comprising administering to an individual in need of such treatment an effective amount of at least one of the peptides as set forth in any one of SEQ ID No. 1-8.

According to a fourth aspect of the invention, there is provided a method of immunizing an individual in need of such treatment against H5N1 infection comprising administering an immunizing amount of at least one of the peptides as set forth in any one of SEQ ID No. 1-8 to said individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Biochemical and function analysis of H5N1-VLP harboring HA cleavage site modification. A. HA, NA and M2 proteins from H5N1 (A/Hanoi/30408/2005) influenza viruses or VSV-G protein were cotransfected with lentiviral vector containing GFP gene and Gag-Pol expressing plasmids, as indicated in material and methods. At 48h transfection, cells were labeled with [³⁵S] methionine for 12 hours. H5N1-VLP was pelleted through 20% sucrose. Purified H5N1-VLPs were divided into two equal parts, and one of them was treated with 3μg of trypsin/ml for 1 h. Labeled H5N1-VLPs and VSV- VLPs were dissolved in RIPA buffer, and viral proteins were precipitated by human anti- influenza serum, rabbit anti-NA and mouse anti-P24 antibodies and analysyzed by PAGE followed by autoradiography of gel. HAO: HA precursor protein; HA1 : HA surface subunit; HA2: HA transmembrane subunit; NA: neuraminidase; CA: HIV capsid protein. B. Dose titration of trypsin for H5N1-pseudotyped lentiviral vectors transduction. Purified H5N1 - VLPs were used to infect 293T cells in the absence or presence of various concentration of trypsin (different). Viral vector titers were determined by P24 ( pg/0.5x10⁵ ). Upper panel: The transduction efficiency, determinated as the GFP-positive cells, was measured by fluorescence-activated cell sorter (FACS) analysis at 48-72hr after infection. Lower panel: The transduction efficiency in 293T cells was also analyzed by PAGE-WB using anti-GFP antibody.

FIG. 2. Both NA and M2 proteins are required for an efficient transduction. A. The lentiviral vectors were pseudotyped with H5N1 HA alone, or with HA+NA, or HA+M2, or HA+NA+M2. The purified vectors titers were quantified by HIV-1 p24 antigen. B. The same amount of viral vectors from A. were used to transduce 293T cells, and 48-72 h post-transduction GFP-positive cells was measured by FACS analysis. C. The lentiviral vector was pseudotyped with H5N1 HA, NA and increasing amount of M2 expressing plasmids. Equal amounts of VLPs were used to infect 293T cells and the expression of GFP protein in transduced cells was analyzed by PAGE-WB using anti- GFP antibody.

FIG. 3. The efficiency of H5N1 -pseudotyped lentiviral vectors transduction differed among cell types. A. The equal amount of p24 antigen H5N1 or VSV-G -pseudotyped lentiviral vectors were used to transduced human embryonic kidney 293T cells, A549 lung carcinoma cell and CD4+ C8166 T cells. The transduction efficiency, determined as the GFP-positive cells, was measured by FACS analysis at 48-72h after infection. B. Fluorescence microscopic images of GFP expression at 48h by H5N1-VLPs applied to 293T and A549 cells.

FIG. 4. NA inhibitor Oseltamivir interfered with H5N1 -pseudotyped lentiviral vectors' transduction in the late and early entry steps. A. HA, NA and M2 proteins from H5N1 influenza viruses or VSV-G protein were cotransfected with an HIV packaging plasmid and transfer vector in 293T cells. 16 hrs later, transfected cells were washed with serum free medium fresh DMEM was added without or with 10OnM Oseltamivir. Pseudotyped VLPs were concentrated from cell free supematants at 48h post- transfection by ultracentrifugation and quantified by HIV-1 p24 antigen. B. The same amount of pseudotyped VLPs (p24) from Oseltamivir treated or non-treated vector producing cells were used to infect 293T cells and the GFP-positive cells were measured by FACS analysis. C. The same amount of H5N1 or VSV-G pseudotyped VLPs (p24) from non- Oseltamivir treated producing cells were used to infect 293T cells in the absence or presence of 10OnM Oseltamivir. The GFP-positive cells were measured by FACS analysis.

FIG. 5. Evaluation of the effects of Oseltamivir on the H5N1 pseudotyped vector transduction by measure the β-gal expression using 96 well plates. Vector pHx'LacZWP encoding for β-gal was co-transfected with H5N1 envelope /VSV-G expression plasmids and HIV packaging plasmid in the absence or presence of oseltamivir. A. A 2x fold dilution of the cultured viral vector stocks were used to transduce 293T cells in 96well plates. 48h post-transduction, the β- galactosidase activity in cell lysates was measured. B. C. Oseltamivir (10OnM) was added to A549 cells in 96wells, followed by adding serial dilution of viral vectors. 48 later, /3-gal positive cells were showed by MAGI assay and counted using Elisapot Reader.

FIG. 6. Bar graph showing inhibition of VLP formation by HA 15 mer peptides. FIG. 7. HA sequence of influenza A/Hanoi/30408/2005 virus.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

As discussed below, the glycoproteins HA and NA were efficiently incorporated onto HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Simultaneous expression of NA and M2 maximized vector production and transduction. Interestingly, the H5N1-pseudotyped HIV vector was sensitive to oseltamivir (tamiflu, an NA inhibitor), amantadine (an M2 inhibitor) or HA peptides independently. This H5N1-pseudotyped HIV vector has been adapted for high-throughput screening of drug candidates such as small molecules, peptides or antibodies which can be safely performed in a biosafety level 2 environment. The H5N1-pseudotyped HIV vector system mimics the influenza virus entry process and can greatly accelerate drug discovery to prepare against the next pandemic.

Described herein is a trypsin-dependent H5N1 HA-pseudotyped lentiviral vector system, which is comprised of plasmids expressing H5N1 HA, NA and M2 membrane proteins and an HIV-based vector expressing a recombinant green fluorescent protein (GFP) or β-Gal. Transduction efficiency was measured quantitatively by FACS analysis or β-Gal activity. This system was evaluated on its transduction efficiency, antiviral sensitivity, and its adaptability for screening antivirals against H5N1 -avian influenza virus surface proteins.

Because of the highly pathogenic features of H5N1 influenza virus, high biosafety containment facilities are required for different H5N1 influenza infection studies, including drug screening experiments. In addition, precautions must be taken when using a wild type H5 hemagglutinin cDNA in the laboratory due to the potential risk of recombination with human influenza viruses. Accordingly, we have developed a H5N1 HA-pseudotyped lentiviral vectors system, in which the cleavage site of H5 was modified to render a "high-pathogenic" H5 to be a "low- pathogenic" form, as being described previously by Li et a/., (Li, Liu et al. 1999) and such modified H5 can only be activated by adding exogenous trypsin. Our data has indicated that that this modified HA was incorporated on the surface of VLPs and it can be cleaved to HA1/HA2 when appropriate amount of trypsin was added (Fig.1 B). Moreover, we demonstrated that, in the absence of trypsin, the HA- pseudotyped VLPs are completely inactivated particles. However, after being activated with exogenous trypsin, these VLPs are capable of efficiently transducing different types of cells (Fig. 1 and 2). Thus, this H5N1 HA-pseudotyped lentiviral vector system is a safe experimental system for study of the avian influenza H5N1 virus entry process. Three influenza membrane proteins of influenza virus, including HA, NA and

M2, play key roles to initiate virus infection. HA protein has been known to be responsible for binding to sialic acid receptors on the cell surface and also mediates fusion of viral membrane and endosomal membrane after endocytosis (Klenk and Rott 1988; Skehel and Wiley 2000). M2 is a small transmembrane protein with an ion channel activity which regulates the pH inside the virion during viral entry cells (Bron, Kendal et al. 1993). The NA has initially been shown to play an essential role in facilitating virus release from infected cells (Palese, Schulman et al. 1974; Palese and Compans 1976; Air and Laver 1989). Recently studies have revealed that NA also contributes to the initial stage of virus infection (Matrosovich, Matrosovich et al. 2004; Ohuchi, Asaoka et al. 2006). Interestingly, two recent studies reported that, in HA-EIAV and HA-MLV vector systems, expression of NA increases the transduction titers of HA-EIAV and HA-MLV vectors (McKay, Patel et al. 2006; Szecsi, Boson et al. 2006). In addition, T McKay, et a/ also showed that presence of M2 augments avian influenza HA pseudotyping (McKay, Patel et al. 2006). Our data further indicates that even though M2 alone did not appear to affect HA-VLPs production, co-expression of this protein with NA significantly enhanced the HA-pseudotyped HIV-based VLPs production (Fig. 2A). Meanwhile, the presence of M2 and NA in HA-pseudotyped VLPs maximized their transduction potential (Fig. 2B and C) All of these results indicate a synergistic effect of NA and M2 proteins at different steps during H5N1 influenza infection and inhibition of each protein's function would significantly disrupt H5N1 infection by affecting virus release and virus entry steps. Accordingly, in a first aspect of the invention, there is provided a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA in the presence of an agent of interest under conditions where in the absence of the agent the influenza HA, M2 and NA would assemble into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles under conditions suitable for infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles wherein an infection rate in the presence of the agent lower than the control infection rate indicates that the agent is a potential anti- influenza agent.

As used herein, cells 'capable of infection refers to any cells known in the art which are capable of or suitable for infection by influenza virus.

As will be appreciated by one of skill in the art, the control does not necessarily need to be repeated each time. In preferred embodiments, additional viral proteins are provided for assembly of the viral particles.

In a preferred embodiment, the HA peptide has been modified such that exogenous trypsin is required is required for HA to be cleaved into HA1 and HA2.

Accordingly, in some embodiments, during the assembly phase, trypsin is added. As discussed herein, this requirement for the addition of trypsin greatly enhances the safety of the recombinant particles.

Accordingly, in another aspect of the invention, there is provided a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins under conditions suitable for assembly into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles in the presence of an agent of interest where in the absence of the agent the virus-like particles would infect the cells capable of infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles.

As will be appreciated by one of skill in the art, control virus-like particles are for example particles grown under similar conditions in the absence of the agent of interest and used to infect cells of the same or similar type, for providing a rate of infection for comparison purposes.

As will be appreciated by one of skill in the art, the rate of infection may be determined by any suitable means known in the art. As discussed herein, in some embodiments, a suitable reporter is used wherein the levels of the reporter are determined or measured.

As will be appreciated by one of skill in the art, this system has several advantages. Specifically, HA, M2 and NA of new viral strains, emerging viral strains or viral strains of concern can easily be substituted for the H5N1 proteins, meaning that potential anti-virals can quickly and easily be screened for effectiveness against a large number of strains. Furthermore, as discussed below, the effectiveness of the anti-virals at the assembly and infection stages can be measured using this system.

In the examples discussed herein, an HIV-based system was used to form the core particles into which HA, M2 and NA were inserted. It is of note that other systems for expressing proteins of interest on the surface of a viral particle known in the art may be used.

As discussed below, the sensitivity of H5N1-pseudotyped HIV vector to an anti-influenza compound was also assessed. The NA inhibitor oseltamivir efficiently inhibited HA-VLPs production (Fig.4A). Results indicated that H5N1- VLPs derived from oseltamivir-treated cells almost completely lost its infectivity, even through equal amounts of VLPs were used to compare the transduction efficiency with VLPs from the non-oseltamivir treated samples (Fig.4B and 5A). In addition, in the absence of NA expression in producer cells, the H5-pseudotyped VLPs exhibited extremely low transduction (Fig. 2B and C). It is clear from these data that NA not only facilitated the release of the VLPs, but also directly affected their transduction ability. It is possible that the NA inhibitor oseltamivir could promote virus aggregation during and/or after virus release, and consequently inhibit viral infectivity (Tai, Escarpe et al. 1998). Moreover, in the H5N1 HA- psedutyped lentiviral vector system, oseltamivir was also found to be capable of interfering with the viral entry step directly (Fig.4C, Fig.5B and 5C). Altogether, it is clear that in this trypsin-dependent H5N1 -psedutyped HIV vector system, three avian influenza membrane proteins act in the same way as they do in the natural H5N1 influenza virus infection and therefore, this system is an ideal system for testing inhibitors against H5N1 membrane proteins or of any influenza strain.

To test the feasibility of this H5N1-pseudotyped HIV vector as a cell-based system for the screening of antivirals, experiments using 96-well plates were also tested. Results showed that after 293T and A549 cells were transduced with a H5N1-VLPs carrying a LacZ gene, the transduction can be easily quantified by using a commercially available β-galactosidase assay kit (CPRG) and/or counting using an Elisapot reader (Fig. 5). Also, in this system, treatment with oseltamivir was shown to effectively inhibit H5N1-VLP's transduction. Therefore, such trypsin- dependent HA-pseudotyped lentiviral vectors system can be used not only for studying viral envelope glycoprotein interactions with their corresponding cellular receptors, but also for identifying compounds having activity against the functions of these avian influenza membrane proteins and the mechanism involved. In addition, this system can be rapidly adapted for monitoring the potential impact of membrane proteins of other highly pathogenic influenza viruses for possible influenza pandemics.

Additionally, we have tested HA 15-mer combinatorial peptide library (108 peptides) for their inhibition effect on H5N1-pseudotyped vector infection in 293T cells. These peptides encompass the full-length heammaglutinin sequence of H5N1 influenza A/Hanoi/30408/2005 virus strain as shown in Figure 7. H5N1-pseudotyped vector was incubated with 293T cells in the presence of

100 μg/ml of each peptide overnight and for 72 hours. The transduced 293T cells were measured by vector-induced β-Gal activity assay. As can be seen in Figure 6, 8 peptides were found to cause more than a 50% reduction in H5N1 -vector induced β-Gal activity. The inhibitory peptides are:

No. 8 MEKNVTVTHAQDILE (SEQ ID NO. 1)

No. 22 CYPGDFNDYEELKML (SEQ ID NO. 2) No. 26 HFEKIQIIPKSSWLS (SEQ ID NO. 3)

No. 39 LWGIHHPNDAAEQTK (SEQ ID NO. 4)

No. 45 LNQRLVPRIATRSKV (SEQ ID NO. 5)

No. 47 TRSKVNGQSGRMEFF (SEQ ID NO. 6)

No. 48 NGQSGRMEFFWTILK (SEQ ID NO. 7) No. 67 TGLRNSPQRERRKKR (SEQ ID NO. 8)

As will be appreciated by one of skill in the art, this clearly shows how the invention can be used in combination with a suitable expression system including a reporter to measure the effects of small molecules, drug candidates and potential therapeutics on viral assembly and transmission. Furthermore, the above-described peptides can be used as direct peptide inhibitors to prevent H5N1 HA infection. For example, an effective amount of any one of a peptide comprising or consisting of or consisting essentially of an amino acid sequence as set forth in any one of SEQ ID Nos. 1-8 or combinations thereof can be administered to an individual in need of such treatment, for example, an individual infected with, suspected of being infected with, at risk of infection with or showing symptoms consistent with infection with H5N1 HA or another influenza strain.

Alternatively, these peptides may be used as vaccines for immunizing individuals in need of such treatment against H5N1 or other influenza strain infection. It is of note that the peptides may be used in the formulation of a vaccine using means known in the art, for example, the HBcAg system in which capsid-like particles are formed which express the peptide of interest on the outer surface of the particles. Similarly, other methods known in the art for increasing antigenicity of small peptides that is recognition of small peptides by a host as foreign may be used.

The H5N1 HA proteins harboring cleavage site modification can be incorporated into HIV-based vector particles and lead to a trypsin-dependent transduction. To establish a H5N1 avian influenza virus entry system, we first constructed three H5N1 membrane protein expressors by synthesizing HA, NA and M2 cDNAs based on H5N1 influenza A virus (A/Hanoi/30408) sequence from database and cloned into a pcGAalpha vector. To reduce the risk of using the wild type H5N1 HA gene, we modified HA cDNA by deleting a five-amino acid sequence in the cleavage site of the HA protein (Fig. 1A). It has been shown that this modification results in an inactivated HA form and requires adding of trypsin to activate it in the experimental condition (Li, Liu et al. 1999). Then, these three H5N1 membrane protein expressors were co-transfected with a HIV-1 based vector pHx'EGFP expressing a green fluorescence protein (GFP) marker and a HIV-1 Gag-Pol-Tat and Rev expressor (Kobinger, 2003) in 293T cells. In parallel, a vesicular stomatitis virus G (VSV-G) glycoprotein expressor was co-transfected with HIV- 1 based vector system as a control. To examine the association of these three H5N1 viral proteins in virus-like particles (VLPs), we radiolabeled the transfected 293T cultures with S³⁵-methionine, and after 16 hours labeling, the supematants were collected and VLPs pelleted from the supernatant through ultracentrifugation. Then, the pelleted VLPs, either treated with trypsin or not, were lysed and immunoprecipitated with corresponding specific primary antibodies, as indicated in figure 1 B. When VLPs were not treated with trypsin, only non-cleaved HA protein (HAO) was detected in VLPs (Fig. 1 B, lane 1 ), whereas for trypsin-treated VLPs, two forms of HA (HA1 and HA2) were detected in the VLP samples (lane 2), indicating that trypsin was able to cleave the modified HA protein. Meanwhile, the NA protein was found in H5N1 pseudotyped VLPs, but not in VSV-G pseudotyped VLPs (Fig. 1 B, middle panel). Both H5N1 and VSV-G pseudotyped VLP show similar level of HIV capsid (p24) protein (Fig. 1 B, lower panel).

To test whether the produced H5N1 HA-pseudotyped VLPs are able to transduce 293T cells and whether this was trypsin-dependent, we incubated 293T cells with similar amounts of HA pseudotyped VLPs (as adjusted by measurement of HIV-1 p24^gag antigen level) in the absence or presence of various concentration of trypsin. After 48 hour of transduction, the percentage of GFP-positive cells was measured by fluorescence- activated cell sorter (FACS) analysis. Results revealed that HA-pseudotyped lentiviral vector particles were only able to efficiently transduce 293T cells in the presence of exogenous trypsin and the optimal efficiency of transduction, about 60% of positive cells, was observed when the cells were treated with 4 μg/ml trypsin (Fig.1 C). Anti-GFP western blot was able to detect abundant GFP protein expression in transduced 293T cells when trypsin was added during the transduction (Fig.1C). Thus, these results indicated that the H5N1 envelop proteins incorporated on VLPs are able to efficiently mediate cell entry and this process is absolutely trypsin-dependent.

Both H5N1 NA and M2 are required for an efficient HA-pseudotyped lentiviral vector particle production and transduction.

Previous studies have shown that the presence of influenza membrane M2 and NA proteins significantly increased FIu-EIAV vector (McKay, Patel et al. 2006) or FIu-MLV vector (Szecsi, Boson et al. 2006) transduction titers. To further investigate the effect of H5N1 NA and M2 proteins on the trypsin-dependent HA-pseudotyped HIV-1 vector system, we transfected 293T cells with HIV-based vector with HA alone or with a combination of HA NA, and M2 expressors, as indicated in figure 3. After 48 hours of transfection, the pseudotyped VLPs were collected and measured for HIV-1 p24 level. Results showed that the VLP production was not affected by expression of M2 alone, while the expression of NA alone only modestly increased VLP production (Fig. 2A). Interestingly, a significant increase of VLP production was observed when both of NA and M2 were expressed during viral production (Fig. 2A), suggesting a requirement of both NA and M2 proteins for a maximal viral production. We next tested the effect of NA and M2 protein on the transduction efficiency of HA-pseudotyped VLPs. To do so, we transduced 293T cells with similar amounts of HA-pseudotyped VLPs (as adjusted by HIV-1 p24 level) and after 48 hours, the transduced (GFP-positive) cells was measured by fluorescence-activated cell sorter (FACS). Results revealed that expression of NA or M2 alone enhanced transduction efficiency by 3 to 5 fold, respectively, however the presence of both NA and M2 lead to a 30-40 x fold increase of transduction in 293T cell lines (Fig.2. B). Meanwhile, the detection of GFP expression in transduced cells also confirmed this observation. Taken together, these results suggest that the expression of both NA and M2 is necessary to maximize the VLP production and transduction efficiency.

The transduction efficiency of H5N1 HA-pseudotyped VLPs for different cell types.

To characterize the tranduction of the H5N1 HA-pseudotyped VLPs in various cell types, different cell lines, including human embryonic kidney 293T cells, A549 lung carcinoma cells and CD4+ C8166 T-lymphoid cells were transduced with similar amounts (as adjusted by VLP-associated p24 levels) of HA-VLPs in the presence of trypsin (4 μg/ml) or VSV-G-VLPs. After 48 hours, each cell population was collected, fixed with 4% paraformaldyhyde and analyzed by FACS analysis. In contrast to VSV-G-VLPs, which transduce all three kinds of cells efficiently, the H5N1 HA-VLPS showed variable transduction ability. For 293T cells, 58% of cells were detected to be GFP-positive, while in A549 and C8166 cells, only 13% and 4% of cell population showed GFP-positive (Fig. 3A). Thus, H5N1 HA-VLPs displayed a cell type-specific transduction ability and the 293T cell is a sensitive cell line for our H5N1 -mediated virus entry system. Given that the transduction of HA-pseudotyped lentiviral vector in epithelium cells is indeed an accurate reflection of the entry steps mediated by the surface proteins of influenza A virus, this trypsin-dependent HA-pseudotyped lentiviral vectors system is not only valuable for studying the mechanism involved in viral entry, but also can be used for screening the anti-viral agents against influenza A virus that target to HA, NA or M2 proteins.

Since the natural primary target for avian influenza is bronchial epithelial cells, we further tested the transduction of H5N1 HA-VLPs for primary bronchial epithelial cells.

NA inhibitor oseltamivir (tamiflu) interfered H5N1-pseudotyped lentiviral vectors' transduction in the late and early entry steps

Previous study has been shown that the M2-inhibitor amantadine inhibited M2 augmentation of transduction of fowl plaque virus HA pseudotyped lentiviral vector based upon equine infectious anemia virus (EIAV) (McKay, Patel et al. 2006). The influenza neuraminidase inhibitor oseltamivir has been shown to block the release of newly formed virus particles and might also interfere with the virus entry step (Matrosovich, Matrosovich et al. 2004; Ohuchi, Asaoka et al. 2006). In this study, we tested whether this trypsin- dependent H5N1 HA pseudotyped HIV-based VLPs is sensitive to influenza specific inhibitors and the effect of oseltamivir on the H5N1 HA pseudotyped VLP production and their transduction in 293T cells. To assess the effect of oseltamivir on VLP production, we introduced oseltamivir in vector producing cells and results showed that adding oseltamivir (100 nM) inhibited HA-VLP production by approximately 3 fold (Fig.4. A). The inhibitory effect on H5N1-VLP production was specific since oseltamivir did not significantly affect the production of VSV-G-pseudotyped vector. To further evaluate the infectivity of the HA-VLPs produced from oseltamivir treated producing cells, an ultracentrifuge was used to remove oseltamivir and purified HA-/ VSV-G-pseudotyped vectors were used to transduce 293T cells. As shown in figure 4B, the H5N1-VLPs derived from oseltamivir treated cells dramatically lost their transduction ability (Fig.4B). As a control, the VSV-G-VLPs from oseltamivir treated cells only had about 1x fold decrease of transduction efficiency. It indicates that oseltamivir not only inhibited VLP production, but also significantly affects their transducing ability. These results are consistent with the previous report by Escarpe et al., suggesting that oseltamivir can promotes virus aggregation (Tai, Escarpe et al. 1998) and therefore disrupt the viral infectivity.

In addition, the direct effect of oseltamivir on the early stage of HA-VLP's transduction was also tested. First, the H5N1 HA-VLPs and VSV-G-VLPs were produced in 293T cells without the treatment of oseltamivir and then, similar amounts of VLPs were incubated with 293T cells in the absence or presence of oseltamivir for 2h at 37 ⁰C. The cell culture was washed with DMEM to remove VLPs and oseltamivir, and cultured in fresh DMEM for 48h. The GFP-positive cells were measured by FACS analysis. As shown in Fig. 4C, oseltamivir reduced the HA-VLPs transduction to 50%, while there was no effect on VSV-G-VLP mediated transduction. All of these results indicate that the NA- inhibitor oseltamivir effectively inhibited H5N1 HA-pseudotyped VLP's activity and meanwhile, proved that this trypsin-dependent HA-pseudotyped lentiviral vector system can be a safe and sensitive system for screening and testing different antivirals against H5N1 HA, NA and M2 proteins as well as against HA, NA and M2 from other influenza strains using the methods described herein.

Potential application of H5N1-pseudotyped lentivial vectors system for large scale testing of anti-H5N1 agents. Given that this H5N1 HA-pseudotyped vector system may provide a safe and sensitive way for testing antivirals against H5N1 membrane proteins, we further set up a quantitative 96-well plate method as a potential screening assay. In this assay, a HIV-1 vector pHx'LacZWP containing a LacZ reporter gene, instead of pHx'EGFPWP, was used to produce HA-VLPs expressing β-galactosidase (β-Gal). These HA-VLPs were then used to transduce 293T or A549 cells in 96 well plates (Fig. 5A and B). After 48h of transduction, the transduction level was quantified by an enhanced β-Gal assay kit (Fig. 5A) or by staining the β-Gal positive cells by a MAGI assay , as previously described (Kimpton and Emerman 1992). The results presented in figure 5A, showed that there was a linear correlation between the input VLPs and the detected β-Gal activity, while for the VLPs produced in the presence of oseltamivir, there was a significantly lower amount of β-Gal activity detected. These results were consistent with the results presented in figure 4B.

Also, the MAGI assay was performed with 96 well plates after A549 cells in each well were transduced by HA-VLPs, and the β-Gal positive cells were scanned and counted (Fig.5B and C). The results indicate that the presence of oseltamivir during transduction reduced the transduction efficiency by 30 to 50% in different viral vector dilutions (Fig. 5B and C). Taken together, this trypsin-dependent HA-pseudotyped lentiviral vectors system can provide a safe and sensitive experimental platform for rapid screening of compounds and/or polypeptides against the H5N1 -avian influenza virus entry step.

Plasmid constructs: The avian influenza H5N1 HA, NA and M2 cDNAs were synthesized based on the sequences of Influenza A/Hanoi/30408/2005 virus from the Influenza Sequence Database. Also, a two-step PCR technique was used to generate a trypsin-dependent H5N1 HA containing a five basic amino acid (RRRKK) deletion and the addition of a threonine residue at proximal to the cleavage site of the protein, as described before (Li, Liu et al. 1999) (shown in Fig 1A). Each of the synthesized HA, NA and M2 cDNA was cloned into a pCAGalpha vector (Watson, Kobinger et al. 2002). The HIV-based vectors encoding for GFP gene (pHxEGFPwp) or encoding for lacZ gene (pHxLacZwp) and the helper packaging construct pCMVΔR8.2 encoding for the HIV helper function, were described previously (Watson, Kobinger et al. 2002). A vesicular stomatitis virus G (VSV-G) glycoprotein expressor was previously described (Yao, Mouland et al. 1998).

Cell culture and transfection: Human embryonic kidney 293T cells and A549 lung carcinoma cell lines were maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin and streptomycin. The CD4⁺ C8166 cells were maintained in RPMI-1640 medium containing 10% FCS and antibiotics. The primary human bronchial epithelial cells were collected from a normal transplant donor and grown in a bronchial epithelial growth media from Cambrex. DNA transfection in 293T cells was performed with CaPO4 precipitation methods as previously described (Yao, Kobinger et al. 1999).

Antibodies and chemicals: Antibodies used in immunoprecipitation are as follows. The rabbit polyclonal to Avian influenza NA protein was purchased from Cedarlane Lab (Hornby, Ontario, Canada). The anti-HIVp24 monoclonal antibody used in this study was previously described (Yao, Mouland et al. 1998). The rabbit anti-GFP was obtained from Molecular Probes Inc. The NA inhibitor Oseltamivir (tamiflu) was obtained from Roch Inc. Trypsin (TPCK-treated) was purchased from Sigma Inc. Enhanced β- galactosidase assay kit (CPRG) was purchased from Genlantis Inc. (San Diego, CA). HIV- 1 p24 ELISA Kit was obtained from the AIDS Vaccine Program of the Frederick Cancer Research and Development Center.

Generation of lentiviral vector and transduction: The pseudotyped HIV-based vector was produced by triple transfection of 293T cells using CaPO4 precipitation methods. Briefly, 293T cells were transfected with the appropriate envelope expression plasmids, the HIV packaging plasmid pCMVΔR8.2 and the HIV-based vector PxEGFPwp or pHxLacZWP (Kobinger, Weiner et al. 2001). After 48 hours of transfection, viral vectors were concentrated from the supernatant through ultracentrifugation, and virus titers were quantified by using RT activity Assay (Yao, Kobinger et al. 1999) or HIV-1 p24 Antigen Capture Assay Kit. Transduction: Cells were plated at 2x10⁴ cells/well in 24-well plate or 4 x10⁵ cells/96 well plates for 24h prior to transduction. The equal amounts of H5N1 or VSV-G- pseudotyped virus-like particles (VLPs) (adjusted by P24 level) or serial dilution (2xfold) of VLPs were incubated with target cells in the 0.5% BSA growth medium containing 4 μg/ml trypsin. After an overnight incubation, transduced cells were replaced with fresh medium. 48h-72h later, the percentage of transduced (GFP-positive) cells was determined using fluorescence-activated cell sorter (FACS) analysis (Becton Dickenson FACS Calibur). The relative transduction efficiency of LacZ-containing vector was evaluated by measuring β-galactosidase in transduced cell lysates using β-galactosidase assay kit (CPRG) by Spectra max plus (Molecular Devices Coporation) or by detecting the GaI positive cells using the MAGI assay as described previously (Kimpton and Emerman 1992). The blue positive cells were counted by Elisapot Reader (AID Autoimmun Diagnostika GmbH). Inhibitor treatment of 293T producer and target cells: 293T cells were plated and transfected with HA, NA and M2 expressors and HIV-based vector as described above. The NA inhibitor Oseltamivir (100 μg/ml) was added to the medium and incubated for additional 24 hours prior to HA-VLPs harvest. To evaluate the effect of oseltamivir on the early stage of HA-VLP's transduction, A549 or 293T cell monolayer cultures in 24-well or 96-well plate were transduced with VLPs in the absence or presence of oseltamivir for

2 hours at 37 ⁰C. Then the cell culture was washed with DMEM to remove remaining oseltamivir, and cultured in fresh DMEM without compound for 48 hours at 37 ⁰C.

Transduced (GFP-positive) cells were analyzed by FACS analysis or the induced β- galactosidase activity in the cells was measured as described above. lmmunoprecipitation analyses. To analyze the VLP-incorporation of HA and NA, HA-VLP-producing 293T cells were starved in methionine-free DMEM for 30 min at

35

48 hours post-transfection and then metabolically labeled with 200 μCi of [ S]- methionine for 16 hours. Labeled viruses were then pelleted, lysed and immunoprecipitated using human anti-flu serum; the supernatant was sequentially immunoprecipitated by rabbit anti-avian influenza NA protein, mouse anti-p24 antibodies. lmmunoprecipitats were then resolved by 10% SDS-PAGE followed by autoradiography. Flourescent microscopy examination, 293T and A549 cells were grown on glass coverslips (12 mm²) in 24-well plate. After 48 hours of transduction, cells on the coverslip were fixed with PBS-4% paraformaldehyde for 5 minutes and were viewed using a confocal microscopy (Olympus IX-70).

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

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Claims

1. A method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA in the presence of an agent of interest under conditions where in the absence of the agent the influenza HA, M2 and NA would assemble into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles under conditions suitable for infection; and comparing the infection rate of said virus-like particles to the infection rate of virus-like particles assembled in the absence of said agent, wherein if the infection rate in the presence of the agent is lower than the infection rate in the absence of the agent, said agent is an anti-influenza agent.

2. A method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA under conditions suitable for assembly into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles in the presence of an agent of interest where in the absence of the agent the virus-like particles would infect the cells capable of infection; and comparing the infection rate of said virus-like particles to the infection rate of virus-like particles incubated with cells in the absence of said agent, wherein if the infection rate in the presence of the agent is lower than the infection rate in the absence of the agent, said agent is an anti-influenza agent.

3. A method of treating an H5N1 influenza infection comprising administering to an individual in need of such treatment an effective amount of at least one of the peptides as set forth in any one of SEQ ID No. 1-8.

4. A method of immunizing an individual in need of such treatment against H5N1 infection comprising administering an immunizing amount of at least one of the peptides as set forth in any one of SEQ ID No. 1-8 to said individual.