WO2009036063A1

WO2009036063A1 - Pseudotyped retroviral vectors and methods of making and using them

Info

Publication number: WO2009036063A1
Application number: PCT/US2008/075853
Authority: WO
Inventors: Gary J. Nabel; Zhi-Yong Yang
Original assignee: The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2007-09-11
Filing date: 2008-09-10
Publication date: 2009-03-19

Abstract

Novel recombinant pseudotyped lentiviral or retroviral vectors comprising a nucleic acid encoding hemagglutinin, a nucleic acidneuraminidase and a nucleic acid a protease capable of cleaving a plurality of HA subtypes to their fusion-active form are useful in a variety of methods including methods for determining the effectiveness of vaccine-mediated immune response, methods for detecting the potency of neutralizing monoclonal antibodies, methods for detecting a compound that affects HA-mediated viral entry into a cell, methods for determining receptor specificity, methods for detecting an influenza infection. In addition, recombinant pseudotyped lentiviral or retroviral vectors comprising a nucleic acid encoding hemagglutinin without a nucleic acid encoding a protease and associated methods are also contemplated.

Description

PSEUDOTYPED RETROVIRAL VECTORS AND METHODS OF MAKING AND USING THEM

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to US provisional application 60/993,378, filed September 11 , 2007, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to recombinant pseudotyped lentiviral and retroviral vectors and associated methods of making and use. Description of the Related Art

Neutralization of influenza HA-mediated viral entry is traditionally measured by the hemagglutination inhibition (HAI) assay. The level of inhibition is considered to correlate with immune protection. The ability to measure viral neutralization and immune protection is an important factor in identifying influenza infection in humans and also in determining the efficacy of commercially available vaccines. The HAI assay has been technically difficult to standardize and there is variability among labs performing the assay due to factors that include variability in virus titers, the quality of the viral preparations, variation in the sources of erythrocytes, and differences in experimental technique from lab to lab. In addition, it is difficult to measure influenza hemagglutinin-mediated (HA-mediated) viral entry by diverse influenza virus HAs and NAs. Thus, there is a need in the art for a sensitive, specific, and standardized assay that measures influenza HA-mediated viral entry by diverse influenza virus HAs and neuraminidases (NAs).

SUMMARY OF THE INVENTION

In response to this need, in one embodiment, a system has been developed to detect or quantify virus neutralization and influenza HA-mediated viral entry by preparing pseudotyped lentiviral or retroviral vectors, that display influenza virus hemagglutinin (HA) and neuraminidase (NA), and include a cellular protease that processes the HA to its fusion- active form. In another embodiment, the system further encodes a reporter, such as a luciferase reporter. In studies conducted on reference sera using this embodiment, this assay was >10 times more sensitive than HAI assays. One embodiment is directed to an assay that is useful in comparing immune responses to different viruses and vaccines. Another embodiment is directed to a system that facilitates the study of viral entry, receptor specificity and natural or vaccine-induced immune responses in many strains of influenza. Other embodiments are useful in measuring the efficacy of the seasonal flu vaccines as well as identifying infection.

More specifically, in one embodiment a recombinant pseudotyped lentiviral or retroviral vector comprises a nucleic acid encoding hemagglutinin (HA), a nucleic acid encoding neuraminidase (NA), a nucleic acid encoding a protease that is capable of cleaving a plurality of HA subtypes to their fusion-active form, and components of a lentivirus or retrovirus, wherein the recombinant pseudotyped lentiviral or retroviral vector displays HA and NA. In another embodiment, the HA in the recombinant pseudotyped lentiviral or retroviral vector does not contain poly basic amino acids at the HA cleavage site.

In another embodiment, the protease is TMPRSS2, such as is human TMPRSS2, a trypsin protease, or a human airway trypsin-like serine protease.

In another embodiment, the HA is influenza HA, such as HA is Hl, H3, H5, H7, or H9, and/or the NA is influenza NA, such as Nl, N2, N3, N7 or N8. In another embodiment the HA is non-H5 influenza HA.

The recombinant pseudotyped lentiviral or retroviral vector described above may further comprise a nucleic acid encoding a reporter, such as a luciferase reporter.

In one embodiment, the recombinant pseudotyped lentiviral or retroviral vector is a recombinant pseudotyped lentiviral vector.

One method is directed to detecting the effectiveness of a vaccine mediated immune response comprising providing a cell in the presence of the recombinant pseudotyped lentiviral or retroviral vector described above and a sample from a vaccinated subject; and detecting neutralization of the recombinant pseudotyped lentiviral or retroviral vector by an antibody, if present, in the sample. In one embodiment, the antibody is labeled. The detecting step may further comprise detecting an amount of neutralization wherein a relatively higher amount of neutralization is indicative of a relatively more effective vaccine or greater immune response. Another method is directed to detecting the potency of neutralizing monoclonal antibodies comprising providing a sample containing monoclonal antibodies in the presence of a cell and the recombinant pseudotyped lentiviral or retroviral vector described above, and detecting the ability of the monoclonal antibody to prevent entry of the recombinant pseudotyped lentiviral or retroviral vector into the cell. The detecting step may comprise detecting the presence of a luciferase reporter inside the cell.

Yet another method is directed to detecting a compound that affects HA-mediated viral entry into a cell comprising providing a cell in the presence of a test sample and the recombinant pseudotyped lentiviral or retroviral vector described above; and detecting recombinant pseudotyped lentiviral or retroviral vector that enters the cell. In one embodiment, the test sample is an antibody or antisera.

A further method is directed to detecting receptor specificity to the recombinant pseudotyped lentiviral or retroviral vector described above comprising contacting a test cell with the recombinant pseudotyped lentiviral or retroviral vector; and detecting whether the recombinant pseudotyped lentiviral or retroviral vector binds with a cell receptor on the test cell; wherein detectable binding indicates the vector has specificity to the test cell.

Another method is directed to detecting an influenza infection in a subject suspected of being infected with influenza virus comprising contacting the recombinant pseudotyped lentiviral or retroviral vector of Claim 12 with a sample from the subject; and detecting binding of the recombinant pseudotyped lentiviral or retroviral vector with an antibody, if present, in the sample. In one embodiment, detectable binding indicates a protective immune response has been raised in the subject.

In the methods described above, the sample may be derived from the vaccinated subject or the sample may be derived from the subject suspected of being infected with influenza virus which is, for example, antiserum or serum. In one embodiment of the methods described above, the detecting is quantitative.

In addition, the methods described above may further comprise subjecting a second sample, test cell, or test compound to the method; and comparing the results of the first and the second sample, test cell, or test compound. Another embodiment is directed to a recombinant pseudotyped lentiviral or retroviral vector comprising a nucleic acid encoding HA, components of a lentivirus or retrovirus, wherein the vector does not comprise a nucleic acid encoding a protease. The recombinant pseudotyped lentiviral or retroviral vector may also further comprise a nucleic acid encoding NA. In one embodiment, the HA is H5 or H7. In another embodiment, the NA is Nl or N7.

This recombinant pseudotyped lentiviral or retroviral vector may be used in a method comprising treating the pseudotyped recombinant lentiviral or retroviral vector with a protease that cleaves HA to its fusion-active form, wherein HA is expressed on the recombinant pseudotyped lentiviral or retroviral vector.

Kits relating to any of the methods described above may include the recombinant pseudotyped lentiviral or retroviral vector, and instructions for use.

Also contemplated is a method of making any of the recombinant lentiviral or retroviral vectors described above comprising co-transfecting cells with the nucleic acid that encodes the HA, the nucleic acid that encodes the NA if present, and the nucleic acid that encodes the protease if present, and components of lentivirus or retrovirus under suitable transfection conditions to produce the expression vector. The components of lentivirus or retrovirus may further comprise a nucleic acid for encoding a reporter, and may include a pCMVΔR8.2 plasmid and a pHR'CMV-Luc plasmid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA-D illustrate the optimization of HA pseudo virus production by addition of neuraminidase (NA). Matched NA was co-transfected with HA to generate pseudotyped lentiviral vectors. Addition of NA (H5N1) enhanced the titer of H5N1 pseudoviruses measured by viral entry with the optimal ratio of HA:NA about 10:1.

Figures 2A-2B illustrate the characterization of H5N1 (KAN- 1/04) pseudotyped lentiviral vectors. Figure 2A illustrates buoyant density gradient analysis of lentiviral vectors. The pseudovirus supernatant was fractioned using Optiprep (Iodixanol) (Yang et al., 2004, J . Virol. 78:5642-5650). The fractions were analyzed for their infectivity, hemagglutination titer against chicken red blood cells (CRBCs), and p24 and H5 protein levels using Western blotting. Peak infectivity corresponded with peak HA titer and peak p24/H5 level in fraction 6 (density 1.12 g/ml, similar to typical lentivirus density (Burtonboy et al., 1993, Arch. Virol. 130:289-300)). Figure 2B illustrates the pH-dependent entry of H5N1 pseudotyped lentiviral vector. The H5N1 or MoMuLV Env (4070A) pseudotyped lentiviral vectors were incubated with indicated amounts of vacuolar type H[+]-ATPase- specific inhibitor, bafilomycin A (Yang et al., 2004, J. Virol. 78:5642-5650). H5N1 pseudoviral entry was inhibited by bafilomycin A in a dose-dependent manner, indicating its entry is pH-dependent, similar to influenza A viruses.

Figures 3A1-A4 and Figures 3B1-B2 illustrate the neutralization of H5N1 pseudoviruses by H5-specific murine monoclonal antibodies (mAb). Figures 3A1-A4 illustrate several H5N1 -pseudotyped lentiviral vectors that were incubated with different amounts of mAbs raised against H5(KAN-l/04) (Yang et al., 2007, Science 317:825-828). Those mAbs showed the best potency against homologous virus, but nevertheless neutralized other H5N1 pseudoviruses with different potency. Figures 3B1-B2 illustrate that the analysis of the same monoclonal antibody against two independent HlNl pseudotyped lentiviral vectors demonstrated no neutralization.

Figures 4A1-A4, 4B1-B2, and 4C illustrate human airway trypsin-like serine protease TMSSPR2 is required in one embodiment for cleavage of HAs lacking poly basic amino acids at the HA cleavage site, and correlation of hemagglutination with the generation of functional HA NA lentiviral vectors. Figures 4A1-A4 illustrate representatives of HlNl, H2N3, H9N2 pseudotyped lentiviral vectors that were produced by cotransfection of the TMPRS S2 membrane serine protease expression vector during the generation of the vector. A marked increase of viral infectivity was observed with the addition of TMPRSS2, indicating its ability to cleave those HAs, which cleavage is essential for HA function (Klenk et al., 1975, Virology 68:426-439; Lazarowitz et al., 1975, Virology 68:440-454). The optimal ratio of HA/NA/TMPRSS2 was -100 ng/12.5 ng/25 ng. Figures 4B1-B2 illustrate that the TMPRSS2 protease is not essential for production of H5N1 and H7N7 pseudoviruses. H5 and H7 contain polybasic amino acids at the HA cleavage site, and thus are subject to cleavage by ubiquitously expressed furin-like protease (Stieneke-Grober, et al., 1992, EMBO J. 11:2407-2414; Van de Ven et al., 1993, Cr it Rev. Oncog. 4:115-136). Figure 4C illustrates that functional H5N1 and H7N7 without protease or HlNl with protease agglutinate CRBCs, are similar to influenza A viruses. HlNl virus was unable to agglutinate CRBCs and yields results similar to the Hl lane.

Figures 5A-D illustrate the specific neutralization of HlNl and H3N2 pseudotyped lentiviral vectors by respective HlNl, H3N2 influenza A reference serum. Neutralization specificity of HA/NA pseudoviruses was demonstrated using influenza A reference sera CDC/Cat. No. VS2400 (HlNl specific) to HlNl(NC/99), H1N1(S1/O6); or reference sera CDC/Cat. No. VS2401 (H3N2 specific) to H3N2(Wyo/03), H3N2(Wis/05) pseudotyped lentiviral vector. IC50 titers of LAI for HlNl (NC/99), H3N2(Wis/05) are 79,767 and 96,916 respectively. Both reference sera have HAI titers of 5120.

Figure 6 illustrates the infection of representative cell lines by HA NA pseudotyped lentiviral vectors. Gene transfer mediated by the indicated H5N1 or HlNl pseudotyped lentiviral vectors was analyzed in the human embryonic kidney epithelial cell line 293A, a primary human foreskin fibroblast (hFib) line, and canine kidney epithelial cell line MDCK. Viral entry was assessed with the luciferase assay as described in the Examples.

Figure 7 is a representation of plasmid VRC7722 (SEQ ID NO: 1) having insert H1(A/New Caledonia/99(H1N1)) (SEQ ID NO: 2), that expresses the Hl protein, GenBank accession number AY289929 ((SEQ ID NO: 3).

Figure 8 is a representation of plasmid VRC7730 (SEQ ID NO: 4) having insert H1(A/South Carolina/18(HlNl)) (SEQ ID NO: 5), that expresses the Hl protein, GenBank accession number AFl 17241 (SEQ ID NO: 6).

Figure 9 is a representation of plasmid VRC9184 (SEQ ID NO: 7) having insert Hl (A/Solomon Island/3/06(HlNl)) (SEQ ID NO: 8) that expresses the Hl protein, GenBank accession number ISDN231558 (SEQ ID NO: 9).

Figure 10 is a representation of plasmid VRC7724 (SEQ ID NO: 10) having insert H3(A/Wyoming/3/03(H3N2)) (SEQ ID NO: 11) that expresses the H3 protein, GenBank accession number AY531033 (SEQ ID NO: 12).

Figure 11 is a representation of plasmid VRC9183 (SEQ ID NO: 13) having insert H3(A/Wisconsin/X-161/05(H3N2)) (SEQ ID NO: 14) that expresses the H3 protein, GenBank accession number ISDN 138723 (SEQ ID NO: 15). .Figure 12 is a representation of plasmid VRC7705(SEQ ID NO: 16) having insert H5(A/Thailand/1(KAN-1)/O4(H5N1)) (SEQ ID NO: 17) that expresses the H5 protein, GenBank accession number AY555150 (SEQ ID NO: 18).

Figure 13 is a representation of plasmid VRC9004 (SEQ ID NO: 19) having insert H7(A/Netherland/219/03(H7N7)) (SEQ ID NO: 20) that expresses the H7 protein, GenBank accession number AY338459 (SEQ ED NO: 21).

Figure 14 is a representation of plasmid VRC9019 (SEQ ID NO: 22) having insert H9(A/Hong Kong/1074/99(H9N2)) (SEQ ID NO: 23) that expresses the H9 protein, GenBank accession number AJ404627 (SEQ ID NO: 24).

Figure 15 is a representation of plasmid VRC9162(SEQ ID NO: 25) having insert Nl (A/New Caledonia/99(H1N1)) (SEQ ID NO: 26) that expresses the Nl protein, GenBank accession number CAD57252 (SEQ ID NO: 27).

Figure 16 is a representation of plasmid VRC9259 (SEQ ID NO: 28) having insert and the N l(A/Brevig_Mission/l/l 8(HlNl)) (SEQ ID NO: 29) that expresses the Nl protein, GenBank accession number AF250356 (SEQ ID NO: 30).

Figure 17 is a representation of plasmid VRC7708 (SEQ ID NO 31) having insert N1(A/Thailand/1(KAN-1)/O4(H5N1)) (SEQ ID NO: 32) that expresses the Nl protein, GenBank accession number AY555151 (SEQ ID NO: 33).

Figure 18 is plasmid VRC9159 (SEQ ID NO: 34) having insert N2( A/Hong Kong/1074/99(H9N2)) (SEQ ID NO: 35) that expresses the N2 protein, GenBank accession number CAB95858 (SEQ ID NO: 36).

Figure 19 is a representation of plasmid VRC9161 (SEQ ID NO: 37) having insert N2(A/Wyoming/3/03(H3N2)) (SEQ ID NO: 38) that expresses the N2 protein, GenBank accession number AAT08001 (SEQ ID NO: 39).

Figure 20 is a representation of plasmid VRC9163 (SEQ ID NO: 40) having insert N7(A/Netherland/219/03(H7N7)) (SEQ ID NO: 41) that expresses the N7 protein, GenBank accession number AARl 1367 (SEQ ID NO: 42).

Figure 21 is a representation of plasmid VRC9267 (SEQ ID NO: 43) having insert N2(A/Wisconsin/67/2005 (NA)) (SEQ ID NO: 44) that expresses the N2 protein, GenBank accession number ABP52004 (SEQ DD NO: 45). If Figure 22 is a representation of plasmid VRC9262 (SEQ ID NO: 46) having insert a Nl (A/Solomon Islands/3/2006(NA)) (SEQ ID NO: 47) that expresses the Nl protein, GenBank accession number ABU99068 (SEQ ID NO: 48).

DETAILEDDESCRIPTION OF THE PREFERRED EMBODIMENT While the development of seasonal influenza vaccines has relied on well-established techniques to assess virus neutralization and vaccine immunogenicity using the hemagglutination inhibition assay (Massicot et al., 1977, J. Infect. Dis. 136 SuppI:S472- S474), it is difficult to standardize the efficacy of vaccines based on antibody responses because of a lack of standardization and variability of results measured in different laboratories (Stephenson et al., 2007, Vaccine 25:4056-4063; Wood et al., 1994, Vaccine 12:167-174). In part, this is due to the variability in virus titers, the quality of the viral preparations, variation in the sources of erythrocytes, and differences in experimental technique from lab to lab. The concerns raised by the recent outbreaks of H5N1 viruses in avian species, its potential for adaptation to humans, and the development of different avian influenza vaccines on an urgent basis have highlighted the need to evaluate the relative potency of alternative vaccine candidates. These concerns underscore the need for improved standardization of immunologic assays to support the assessment of vaccines advancing in development. It is well known that the main target for virus neutralization is the viral hemagglutinin (HA) (Virelizier, J. L., 1975, J. Immunol. 115:434-439). Antibodies directed to this target can neutralize the virus through interactions with one or more of five major sites on the globular head of HA previously defined by serologic and structural techniques (Gerhard et al., 1981. Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. Nature 290:713-717; Raymond et al., 1986, Virology 148:275-287; Skehel et al., 2000, Annu Rev Biochem. 69:531-569; Wiley et al., 1981, Nature 289:373-378; Wilson et al., 1981, Nature 289:366-373). Recently it has become possible to use molecular genetic techniques to express the viral hemagglutinin on the surface of lentiviral vectors, which can then be used for gene transfer (Kong et al., 2006, Proc. Natl. Acad. ScL USA 103:15987-15991). Through the use of luciferase or other reporter genes, it is then possible to quantitate the degree of viral entry, and this assay has been successfully used for avian influenza viruses with a polybasic amino acid sequence at the protease cleavage site and for the 1918 influenza virus HA with such modifications inserted at this protease site (Kong et al., 2006, Proc. Natl. Acad. ScL USA 103:15987-15991).

At the same time, it has proven difficult to develop pseudotyped lentiviral vectors for all influenza virus strains, which could greatly advance an understanding of viral entry among different strains, allow additional analysis of the mechanism of attachment of influenza virus, and facilitate comparisons of influenza virus vaccine immunogenicity among laboratories. Given the importance of NA and various proteases in generating replication-competent influenza A viruses, a systemic approach was taken to investigate their role in generating functional HA/NA pseudovirus.

The ability to analyze influenza virus entry has been confined largely to studies of intact virus, which has been difficult until recently to analyze using molecular genetic tools (Stevens et al., 2006, Nat. Rev Microbiol. 4:857-864). More recently, it has been possible to generate recombinant influenza viruses using reverse genetics, which has proven to be a major advance in the development of vaccine strains with novel serotypes (Hoffmann et al., 2000, Proc. Natl. Acad. ScL USA, 97:6108-6113; Neumann et al., 1999, Proc. Natl. Acad. ScL US A. 96:9345-9350; Webby et al., 2004, Lancet 363:1099-1103). However, the analysis of HAI and viral entry in these systems is limited by the necessity of growing these viruses in producer cells, the requirement of a higher containment facility for lethal viruses, and their ability to mediate gene transfer (Latham et al., 2001, J. Virol. 75:6154-6165). Though readily performed, this assay varies among laboratories because of variations in virus preparations and erythrocyte target cells, making comparisons among laboratories difficult. Thus, there is a need in the art to develop a sensitive, specific, and standardized assay that measures entry by diverse influenza virus HAs and NAs that will facilitate such comparisons and accelerate the generation of vaccines, monoclonal antibodies and antiviral drugs targeted to HA and NA.

Previously, it has been possible to use murine retroviruses or lentiviral vectors to develop pseudotyped reporter viruses that allow the further analysis of viral entry, to define receptors, and to isolate neutralizing antibodies to further study their function (Li et al., 2003, Nature 426:450-454; Yang et al., 1998, Science 279:1034-1037; Yang et al., 2004, J. Virol. 78:5642-5650). While this approach has proven useful in the study of a number of viruses, and it has been recently accomplished for H5N1 influenza virus pseudotypes (Kong et al., 2006, Proc. Natl. Acad. ScL USA 103:15987-15991; Temperton et al., 2007, Influenza and Other Respiratory Viruses 1:105-112), it was not possible to generate viral pseudotypes for diverse Hl and H3 influenza virus strains.

In one embodiment of the present invention, such pseudotyped lentiviral or retroviral vectors are generated using a combination of hemagglutinin, neuraminidase and a cellular protease required to develop the active form of the viral spike. Gene transfer is observed in several different cell lines, including those readily infected by influenza viruses as described in PCT/US2007/081002, which is incorporated herein by reference. The availability of this tool to analyze viral entry facilitates the study of lentiviral or retroviral vectors in a variety of ways. In another embodiment, lentiviral or retroviral vectors can also mediate hemagglutination, provided that a relevant NA and the protease are included for strains where they are required to generate functional lentiviral or retroviral vectors. Without being bound by theory, it appears that hemagglutination correlates with the ability to mediate entry and that HAI likely indirectly measures inhibition of functional viral spikes.

In one embodiment, a combination of the viral hemagglutinin together with neuraminidase and a cellular protease, such as TMPRSS2 (Bottcher et al., 2006, J. Virol. 80:9896-9898), permits the generation of lentiviral or retroviral vectors from all influenza virus strains analyzed. Without being bound by theory, this finding suggests that TMPRSS2 may be involved in the processing of a variety of influenza HAs, and this approach facilitates an understanding of influenza virus entry while providing a high-throughput, sensitive and quantitative measure of neutralizing antibody titer for vaccine development, therapeutic monoclonal antibody production, and further analysis of host-virus interactions.

Among the proteases tested for their ability to generate functional HA NA lentiviral vector pseudotypes, in one embodiment, in this example the human membrane serine protease TMPRS S2 was able to generate functional lentiviral vectors, even though both serine proteases TMPRS S2 and HAT (human airway trypsin-like serine protease) have been reported effective in cleaving HAs on influenza viruses previously (Bender et al., 1999, Virology 254:115-123). Without being bound by theory, this difference could result from the differences in trafficking of lentivirus and influenza viruses or cell type differences related to the use of producer cells. In either case, TMPRSS2 is implicated in one embodiment as a protease relevant to processing of a variety of influenza virus HAs.

In this embodiment, an assay provides a method for analyzing the potency of neutralizing antibodies developed by vaccination or through screening of neutralizing monoclonal antibodies. This embodiment has been used to screen for monoclonal antibodies capable of neutralizing H5N1 and relatively broadly neutralizing antibodies that are useful for diagnostic and therapeutic purposes were identified (Yang et al., 2007, Science 317:825-828). In another embodiment, a similar approach is used to analyze such antibodies for alternative influenza strains. Compared to the traditional HAI assay and microneutralization (Rowe et al., 1999, J. Clin. Microbiol. 37:937-943), the LAI demonstrated increased sensitivity relative to the traditional assay using CDC reference sera (Fig. 5, legend). In another embodiment this assay is standardized and validated and used to analyze sera from a variety of different sources and serves as a comparison for the potency of different vaccines analyzed in alternative trials. Such a comparison facilitates the identification of the most potent immunogens and adjuvants required to stimulate optimal immunity for an avian influenza virus and other pandemic or seasonal infections. Based on the similarity between the HA NA pseudotyped lentiviral or retroviral vectors and influenza A viruses in entry and hemagglutination, this system therefore provides fundamental insight into the mechanisms of viral entry and the specificity of HA for its receptor. One embodiment also provides a platform assay for quick and effective study of the adaptation of any HA from avian influenza virus having potential to become human pandemic flu, as recently described (Yang et al., 2007, Science 317:825-828). This analysis facilitates comparisons of alternative influenza vaccine candidates and adjuvants, allowing more effective responses to influenza virus outbreaks.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. In one embodiment, viral vectors (e.g., replication defective retroviruses or lentiviruses) serve equivalent functions.

A pseudotyped lentiviral or retroviral vector is a replication defective lentiviral or retroviral vector, also called pseudoviruses, wherein the envelope protein is exchanged for a viral spike from HA, such as influenza HA. In one embodiment, a pseudotyped lentiviral or retroviral vector is made from components of a lentivirus, such as HIV, or from components of a retrovirus, such as murine retrovirus. These vectors may enter a cell much like the virus, for example influenza virus, from which the HA is derived enters a cell. In one embodiment, HA and NA proteins are displayed on (expressed on the surface of) the pseudotyped lentiviral or retroviral vector. In another embodiment, the vector delivers a reporter gene to measure gene transfer. For example, in one embodiment, a pseudotyped lentiviral or retroviral vector may be prepared using a packaging vector such as pCMVΔR8.2 plasmid and a transfer vector such as pHR'CMV-Luc plasmid.

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to DNA encoding HA, NA, and a cellular protease.

As used herein, a "recombinant" pseudotyped lentiviral or retroviral vector is a vector wherein the material (e.g., a nucleic acid or protein) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. Specifically, e.g., a protein derived from influenza virus is recombinant when it is produced by the expression of a recombinant nucleic acid. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, or other procedures, or by chemical or other mutagenesis; and a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein which is produced by expression of a recombinant nucleic acid. One embodiment of a recombinant nucleic acid includes an open reading frame encoding an HA, NA, and/or a protease, and can further include non-coding regulatory sequences, and introns.

Recombinant pseudotyped lentiviral or retroviral vectors include nucleic acids encoding HA and optionally NA and/or a cellular protease. In one embodiment, the HA and NA are derived from influenza A viruses, which are enveloped negative single-stranded RNA viruses that infect a wide range of avian and mammalian species. Influenza A viruses are classified into serologically-defined antigenic subtypes of the HA and NA major surface glycoproteins. Table 1 shows hemagglutinin subtypes of influenza A viruses isolated from humans, lower mammals and birds. Nucleic acids encoding these HA subtypes are useful in embodiments of the present invention. Table 1

aThe reference strains of influenza viruses, or the first isolates from that species, are presented. bCurrent subtype designation. From WHO Memorandum 1980 Bull WHO 58:585-591.

In one embodiment, nucleic acids encoding Hl, H3, H5, H7, or H9 are used. In another embodiment, nucleic acids encoding HA do not include H5. NA is encoded on a separate RNA molecule. NA cleaves terminal sialic acid residues of influenza A cellular receptors and is involved in the release and spread of mature virions. It may also contribute to initial viral entry.

Nucleic acids may be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single- stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

In one embodiment, recombinant pseudotyped lentiviral or retroviral vectors include a nucleic acid encoding a cellular protease that processes HA into its fusion active form. Examples of cellular proteases useful in an embodiment of the invention include transmembrane protease serine 2 (TMPRSS2), such as human TMPRSS2, a trypsin protease, or a human airway trypsin-like serine protease. In one embodiment, recombinant pseudotyped lentiviral or retroviral vector that displays H5 or H7, such as a vector that displays H5N1 or H7N7, does not contain a nucleic acid encoding a cellular protease.

As used herein, the terms "detect", "detecting", or "detection" may describe either the general act of discovering or discerning or the specific observation of a detectably labeled compound or composition, such as a labeled antibody. Detecting neutralization or binding refers to qualitative or quantitative detection. Thus, detecting includes detecting the presence or absence of neutralizing or binding of a psuedotyped lentiviral or retroviral vector or pseudovirus by an antibody. In addition, in one embodiment, detecting also includes detecting the quantity of antibodies that bind to psuedotyped lentiviral or retroviral vectors or pseudoviruses. In another embodiment, detecting includes detecting the quantity of cells that exhibit the presence of reporter, for example a luciferase reporter, inside the cell.

Detection can also be accomplished using any of a variety of other immunoassays. The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as i₅₂Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Examples of bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

In one embodiment, detecting comprises detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase). Following introduction of the expression construct into the cells, expression of the reporter gene can be determined by conventional means. Any assay which detects a product of the reporter gene, either by directly detecting the protein encoded by the reporter gene or by detecting an enzymatic product of a reporter gene-encoded enzyme, is suitable for use in the methods described herein. In one embodiment, a reporter gene is included in a transfer vector, such as pHR'CMV-Luc plasmid, used in making recombinant pseudotyped lentiviral or retroviral vector. Thus, in one embodiment, cell entry is measured by a luciferase assay.

Cells and test cells are used in the methods and assays of the invention. In one embodiment, a cell is characterized by its ability to be readily infected with a specified virus. In another embodiment, a cell is capable of allowing entry of a specified virus into the cell, such as an influenza virus. A cell derived from human embryonic kidney cell lines 293 A and 293T, primary human foreskin fibroblast (hFib) line, and canine kidney epithelial cell line MDCK are useful in methods described herein.

In another embodiment, a test cell is one that is suspected of being infected with a specified virus or at least that is capable of allowing entry of a specified virus into the cell, such as an influenza virus.

Viral entry is the earliest stage of infection in the viral life cycle, as the virus, or recombinant pseudotyped lentiviral or retroviral vector, comes into contact with a cell and introduces viral material into the cell. HA displayed on a virus or a recombinant pseudotyped lentiviral or retroviral vector, for example, mediates viral entry into a cell by attaching to a receptor on the cell. A receptor is a protein molecule, embedded in a part of a cell, to which HA may attach. Influenza HA, for example, binds to sialic acid (SA) cell reporters in the respiratory tract. Subsequently, membrane fusion occurs where the cell membrane is punctured, an entry pore is formed, and the virus or vector ultimately penetrates the cell. In one embodiment, similar to replication-competent influenza viruses, entry mediated by the recombinant pseudotyped lentiviral or retroviral vector is sensitive to an endosomal inhibitor, bafilomycin A, indicating the vector's entry is pH-dependent, similar to influenza A viruses. In one embodiment of a method of the invention, specificity of HA displayed on a recombinant pseudotyped lentiviral or retroviral vector to a receptor in a test cell may be studied.

In an embodiment of a method of the invention, monoclonal antibodies are used. Monoclonal antibodies are well known in the art and may be prepared using methods known in the art. In one embodiment, murine monoclonal antibodies are used in methods of the invention. In another embodiment, animals are immunized against a virus such as influenza, for example with a DNA prime, adenovirus boost vaccine, to prepare monoclonal antibodies.

"Subject" refers to any member without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The assays described above are intended for use involving any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.

A sample from a vaccinated subject or an infected subject includes any sample taken from the subject that is capable of containing antibodies raised by an immune response from a vaccine or infection such as an influenza vaccine or an influenza infection. In one embodiment, a sample is a bodily fluid such as blood, anti-serum, serum, urine, or broncoalveolar lavage fluid.

An infected subject is a subject that has been exposed to a virus such as influenza that causes a natural immune response in the subject. A vaccinated subject is a subject that has been administered a vaccine that is intended to provide a protective effect against a virus such as influenza.

Neutralization refers to binding of antibodies to an antigen such as the recombinant pseudotyped lentiviral or retroviral vector or pseudovirus and affects the antigen activity, such as activity relating to preventing infection. In one embodiment, neutralization prevents the recombinant lentiviral or retroviral vector or pseudovirus from entering a cell. Binding of antibodies to the recombinant lentiviral or retroviral vector or pseudovirus does not necessarily require an effect on the activity.

An "immune response" to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. For purposes of the present invention, a "humoral immune response" refers to an immune response mediated by antibody molecules, including secretory (IgA) or IgG molecules, while a "cellular immune response" is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen- specific response by cytolytic T-cells ("CTL"s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+T-cells. In addition, a chemokine response may be induced by various white blood or endothelial cells in response to an administered antigen.

Thus, an immunological response as used herein may be one that stimulates CTLs, and/or the production or activation of helper T-cells. The production of chemokines and/or cytokines may also be stimulated. The antigen of interest may also elicit an antibody- mediated immune response. Hence, an immunological response may include one or more of the following effects: the production of antibodies (e.g., IgA or IgG) by B-cells ; and/or the activation of suppressor, cytotoxic, or helper T-cells and/or T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.

One embodiment of the invention is directed to kits. The kits are useful in assays for detecting the effectiveness of a vaccine mediated immune response, detecting the potency of neutralizing monoclonal antibodies, detecting a compound that affects each HA-mediated viral entry into a cell, detecting receptor specificity to a recombinant lentiviral or retroviral vector, and/or detecting influence the infection. For example, the kit may include a recombinant lentiviral or retroviral vector, and one or more of cells, tests cells, antibodies, for example labeled antibodies, and/or a standard(s) such as reference sera. The recombinant lentiviral or retroviral vector and other components can be packaged in a suitable container(s). The kit may further comprise instructions for using the kit in accordance with methods described herein.

Vector nucleic acids can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989), and other laboratory manuals. Transfection conditions are conditions such as those mentioned above that are used in transfection techniques.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Other selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding HA, NA, and a cellular protease or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

EXAMPLES

Example 1

The following materials and methods were used in Example 1.

Cell lines, media, and antibodies

Human embryonic kidney cell lines 293A and 293T were purchased from Invitrogen (Carlsbad, CA) and were maintained in DMEM (Invitrogen) containing 10% FBS (Sigma, St. Louis, MO). Anti-Hl, H5, H7, H9 rabbit polyclonal antibodies were obtained from Immune Technology (New York, NY). Hl and H3 reference sera were kindly provided by Dr. Alexander Klimov from the Centers for Disease Control, U.S.A.

Genes, expression vectors, and HA gene expression

Genes encoding H1(A/New Caledonia/99(H1N1)), H1(A/South Carolina/18(H IN I)), H1(A/Solomon Island/3/06(HlNl)), H3(A/Wyoming/3/03(H3N2)), H3(A/Wisconsin/X- 161 /05(H3N2)), H5(A/Thailand/1 (KAN- 1 )/04(H5N I)), H7(A/Netherland/219/03(H7N7)), H9(A/Hong Kong/1074/99(H9N2)) (GenBank accession nos. AY289929, AFl 17241, ISDN231558, AY531033, ISDN138723, AY555150, AY338459, and AJ404627 respectively), and N1(A/New Caledonia/99(H1N1)), Nl(A/Brevig_Mission/l/18(HlNl)) N1(A/Thailand/1(KAN-1)/O4(H5N1)), N2(A/Hong Kong/1074/99(H9N2)),

N2(A/Wyoming/3/03(H3N2)), N7(A/Netherland/219/03(H7N7), N2(A/Wisconsin/67 2005), and N1(A/Solomon Island/3/2006)) (GenBank accession nos. CAD57252, AY555151, CAB95858, AAT08001, AARl 1367, ABP52004 and ABU99068 respectively) and were synthesized using human-preferred codons by GeneArt (Regensburg, Germany) and were subsequently cloned into expression vector CMWR (Barouch et al.). Expression of the HAs was also confirmed by Western blotting analysis. Exemplary plasmids having the HA and NA gene inserts described above are shown in Figures 7-22.

Pseudovirus production

The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described (Naldini et al., 1996, Proc. Natl. Acad. Set. USA 93:11382- 11388; Yang et al., 2004, J. Virol. 78:5642-5650). Briefly, 293T cells in a 6-well- dish were co-transfected with the indicated amounts of HA, NA, TMPRSS2 along with 1.2 μg of pCMVΔR8.2 and 1.2 μg of pHR'CMV-Luc plasmid using a calcium phosphate transfection kit (Invitrogen, Carlsbad, CA) overnight, and replenished with fresh media. 48 hours later, supernatants were harvested, filtered through a 0.45 μm syringe filter, stored in aliquots, and used immediately or frozen at -8O⁰C. The input viruses were standardized by the amount of p24 in the virus preparation. The p24 level was measured from different viral stocks using the HIV-I p24 Antigen Assay kit (Beckman Coulter, Fullerton, CA). Analysis of HA expression in these preparations was confirmed after buoyant density centrifugation using Western blot analysis, and levels varied by no more than 2-fold.

Viral infection and luciferase assay

A total of 30,000 293A cells were plated into each well of a 48-well dish one day prior to infection. Cells were incubated with 100 μl of viral supernatant/well in duplicate with HA-pseudotyped viruses for 4-16 hours. Viral supernatant was replaced with fresh media at the end of this time, and luciferase activity was measured 24-48 hours later as previously described (Yang et al., 2004, J. Virol. 78:5642-5650) using "mammalian cell lysis buffer" and "luciferase assay reagent" (Promega, Madison, WI) according to the manufacturer's protocol.

Hemagglutination assays

Hemagglutination of chicken RBC (CRBC) was done as previously described (Glaser et al., 2005, J. Virol. 79:11533-11536; Paulson et al., 1987, Methods Enzymol. 138:162-168; Yang et al., 2007, Science 317:825-828). In brief, freshly prepared CRBCs (Innovative Research, Southfield, MI) were washed three times with 10 ml PBS (pH 7.4). To measure the binding activity of pseudoviruses by hemagglutination, 50 μl of 1 :5 diluted H5N1 pseudovirus in PBS were added to a 96-well round bottom plate and serially diluted two-fold. 50 μl of 0.5% washed CRBC were added respectively, and mixed with virus. HA titers were determined 60 min. later by visual inspection.

Neutralization assay

HA-pseudotyped lentiviral vectors encoding luciferase were first titrated by serial dilution. The concentration of viruses giving 25% maximum activity (p24 = 12.5 ng/ml) was then incubated with indicated amounts of antiserum or monoclonal antibodies for 20 minutes at room temperature and added to 293A cells (30,000 cells/well) (120 μl/well, in triplicate). Plates were washed and replaced with fresh media 4-16 hours later. Luciferase activity was measured after 24-48 hours.

Generation of antisera

Female BALB/c mice, 6-8 weeks old (Jackson Labs), were immunized as previously described (Kong et al., 2006, Proc. Natl, Acad. Sci. USA 103:15987-15991). Briefly, mice were immunized three times with 15 μg plasmid DNA in 100 μl of PBS (pH 7.4) intramuscularly at weeks 0, 3, 6 for DNA immunization alone, or for prime-boost vaccination to generate neutralizing monoclonal antibodies, followed by additional boosting with 10¹⁰ particles of recombinant adenovirus (rAd) expressing the same antigen at week 10. Serum was collected 10 days after the last vaccination. Animal experiments were conducted in full compliance with all relevant federal guidelines and NIH policies.

Buoyant density gradient analysis of lentiviral vectors

Pseudovirus supernatants were harvested and filtered through a 0.45-μm syringe filter. The recombinant virus was first concentrated by layering a 10-ml sample in tissue culture media onto 3 ml of Optiprep (Iodixanol) (60%) solution (Invitrogen, Carlsbad, CA) and centrifuging it at 53,000 x g for 1 h in a Sorvall TH-641 rotor (Kendro, Newtown, CT). The top 7 ml of supernatant was removed, and the remaining solution was mixed uniformly to achieve a final concentration of 30% Optiprep in a 6-ml final volume. The gradient was formed by centrifugation at 363,000 x g for 3.5 h with a Beckman NVT-100 rotor. Fractions of -0.5 ml were collected and analyzed for viral entry, hemagglutination activity, and for p24 and HA level by Western blotting. pH-dependent entry of influenza A hemagglutinin-pseudotyped lentiviral vectors

293A cells were plated in a 48-well dish (30,000 cells/well) the day before infection. Cells were preincubated with the indicated amounts of bafϊlomycin A (Sigma) for 1 h. Pseudoviruses were mixed with the same concentrations of reagents in tubes and added to cells. Two hours later, viruses were removed and replaced with fresh medium. Cells were harvested 48 h after infection, and a luciferase assay was performed.

Statistical analyses

The correlation between HAI and LAI (IC50) was examined using a rank-based Spearman's rho. Linear regression was used to determine whether the relationship between HAI and LAI (IC50) was independent of dose level. Since this model showed no evidence of either a main effect of dose or an interaction between dose and LAI (IC50) in modeling HAI, the rank-based correlation as measured by Spearman's rho was used as a primary measure of association between the two assays.

The results of the experiments above are discussed below in detail

Co-expression of NA with HA enhances pH-dependent gene delivery by H5N1 but not other pseudotyped lentiviral vectors

We have previously shown that the avian influenza virus hemagglutinin can mediate gene transfer when used as a pseudotype on lentiviral vectors (Kong et al., 2006, Proc. Natl. Acad. ScL USA 103:15987-15991). We next evaluated whether the addition of the viral neuraminidase could affect gene transfer by this vector. Co-transfection of increasing quantities of the neuraminidase revealed a dose-responsive increase in gene transfer, which enhanced viral entry more than 10-100 fold (Fig. 1; H5N1, left panel). It was previously known that the H5 HAs could readily pseudotype lentiviral vectors, but this was not highly effective for non-H5 strains (Kong et al., 2006, Proc. Natl. Acad. Sci. USA 103:15987-15991; Temperton et al., 2007, Influenza and Other Respiratory Viruses 1:105-112). To compare the effect of NA on non-H5 viral HAs, a matched NA expression vector was cotransfected with Hl and H3 HAs. Minimal effects on gene transfer were observed, and it remained difficult to generate effective HA NA-pseudotyped lentiviral vectors (Fig 1 ; HlNl and H3N2 panels).

The ability of these viral hemagglutinins to incorporate into the lentiviral particle was confirmed by analysis using buoyant density gradient fractionation with H5N1 pseudotyped lentiviral vectors. The individual fractions of increasing buoyant density were prepared using Opti-PrepR, used previously to fractionate lentiviral vectors (Yang et al., 2004, J. Virol. 78:4029-4036). Gene transfer activity in individual fractions correlated with the viral hemagglutination titer in these fractions (Fig. 2A, upper panel). In addition, the virion- associated HA was also detected by Western blotting (Fig. 2A, lower panel) and was present mainly as the cleaved product, similar to its form in H5N1 influenza virus (2). Similar to replication-competent influenza viruses, entry mediated by the HA NA lentiviral vectors was sensitive to an endosomal inhibitor, bafilomycin A, in contrast to the pH-independent Moloney murine leukemia virus (MoMuLV) glycoprotein pseudotyped vector (Fig. 2B). Neutralization of HANA pseudotyped lentiviral vectors by HA-specific antibodies To confirm the role of HA in viral entry and to document its sensitivity to antibody neutralization, inhibition studies were performed. Antisera were raised by DNA immunization of mice against the H5 (KAN-I) HA. Such immune sera have been shown previously to inhibit the replication of live virus in the microneutralization assay and are active in the viral hemagglutination inhibition assay (Kong et al., 2006, Proc. Natl Acad. Sci. USA 103:15987-15991; Yang et al., 2007, Science 317:825-828). In addition, animals immunized with a DNA prime, adenovirus boost were used to prepare monoclonal antibodies that could also mediate inhibition of viral entry (Yang et al., 2007, Science 317:825-828). Incubation of several related avian H5N1 pseuodotyped lentivirus vectors with monoclonal antibodies specific for H5N1, 11H12 and 9E8 (Yang et al., 2007, Science 317:825-828), resulted in a marked dose-dependent decrease in viral gene transfer (Fig. 3A). In contrast, HlNl lentiviral vectors showed no evidence of neutralization by this monoclonal antibody (Fig. 3B). These data indicated that lentiviral entry was dependent on the viral HA and documented its specificity.

The cellular serine protease TMPRS S2 allows generation of functional HA NA lentiviral vectors from influenza strains lacking a polybasic cleavage site

We have shown previously that modification of a cleavage site in HA could improve its ability to pseudotype using the 1918 HlNl influenza virus strain (Kong et al., 2006, Proc. Natl. Acad. Sci. USA 103:15987-15991). This finding suggested that the appropriate proteolytic cleavage could affect the activity of such pseudotyped viruses; however, it remained difficult to achieve efficient entry, and limited efficacy was achieved with other HlNl viruses. To address this problem, we first attempted treatment with trypsin, as is often done to activate influenza viruses. Recently, two cellular serine proteases, TMPRSS2 and human airway trypsin-like protease (HAT), were reported to cleave a variety of influenza HAs (Bottcher et al., 2006, J. Virol. 80:9896-9898). To determine whether these proteases could provide appropriate proteolytic cleavage and processing to generate functional HA NA lentiviral vectors, increasing amounts of expression vectors each were co-transfected with the HlNl expression vectors, and pseudovirus entry was measured. Inclusion of the TMPRS S2 expression vector, together with HA and NA, during the generation of lentiviral vectors increased gene delivery more than four orders of magnitude for two HlNl strains (Fig. 4A, HlNl (NC/99) and HlNl (SC/18)) under optimal conditions. This effect was not seen in the absence of NA, suggesting that in addition to cleavage by TMPRSS2, in one embodiment, NA is essential for virion release and HA competence through its ability to trim sialic acid (SA) on HA, as observed in influenza A infection (Palese et al., 1974, Virology 61:397-410). In addition, a similar effect was observed for two other viral strains (Fig. 4A, H3N2 (Wyo/03) and H9N2 (HK/99)). The inclusion of TMPRSS2 had minimal effect on the generation of functional pseudotyped lentiviral vectors for strains with polybasic amino acids in the cleavage site, such as H5N1, as shown previously (Bottcher et al., 2006, J. Virol. 80:9896-9898) and H7N7 (Net/03) (Fig. 4B). No increase in pseudotype reporter activity was observed by co-expression of HAT and HA with or without NA. Therefore, combined co-transfection of the serine protease TMPRS S2 with HA and NA effectively produced pseudotyped lentiviral vectors derived from diverse influenza virus strains capable of mediating viral entry regardless of the nature of their cleavage sites. It is expected that other cellular proteases that are capable of clearing a plurality of HA subtypes to their fusion- active form will also be effective.

Functional HA NA pseudotvped lentiviral vectors mediate hemagglutination with chicken erythrocytes: requirement for NA

To compare the lentiviral vectors to influenza viruses further, we evaluated their ability to hemagglutinate chicken red blood cells (CRBCs) by assessing the HA, NA, and processing requirements for this interaction to occur. Although H5 pseudotyped viruses could mediate gene transfer (Kong et al., 2006, Proc. Natl. Acad. ScL USA 103:15987- 15991), no viral hemagglutination was achieved (Fig. 4C, H5). In contrast, when NA was cotransfected to generate the pseudotyped virus, this hemagglutination was readily observed (Fig. 4C, H5N1). Similar results were observed with H7 and H7N7 (Fig. 4C). In contrast, for HlNl, pseudotyped lentiviruses did not cause hemagglutination unless the TMPRSS2 expression vector was used to generate the pseudovirus (Fig. 4C, Hl vs. HlNl + TMPRSS2 and legend). These findings suggest that the ability to cause hemagglutination is associated with the generation of functional lentiviral vectors and that these pseudotyped vectors recapitulate the ability of natural influenza virus to undergo viral hemagglutination. Thus, it is likely that the physiologic structures found on the influenza virus can be recapitulated on the pseudotyped lentiviral vector.

Neutralization specificity and sensitivity of HA NA pseudotvped lentiviral vectors: comparison of HAI and LAI (lentiviral assay inhibition) with reference and human vaccine sera

Finally, we examined the ability of HA NA lentiviral vectors to measure virus neutralization using reference antisera and sera from human subjects of influenza vaccine clinical studies. Reference antisera obtained from the Centers for Disease Control showed specificity for recent HlNl and H3N2 influenza viruses. These sera showed high comparable specificity for the related, but not unrelated, HA NA lentiviral vectors at high titer (>1 : 10,000) as measured by the reduction in luciferase reporter activity relative to a negative control (Fig. 5). Neutralization was also assessed in sera from human subjects vaccinated with an inactivated H5N1 experimental vaccine, in whom HAI titers had been previously measured (Treanor et al.). A highly significant association was found between the result measured by LAI and by HAI (Table 1, rho=.87, p<0.0001). In addition, the sensitivity of LAI was approximately ten-fold higher than the HAI assay, and in some cases, vaccine responses could be detected in subjects that were considered unresponsive in the HAI assay. These data show that TMPRSS2 can support the processing of HAs from diverse influenza strains and document the sensitivity and specificity of the LAI assay, as well as its correlation with traditional HAI measurements.

Claims

WHAT IS CLAIMED IS:

1. A recombinant pseudotyped lentiviral or retroviral vector comprising a nucleic acid encoding hemagglutinin (HA), a nucleic acid encoding neuraminidase (NA), a nucleic acid encoding a protease that is capable of cleaving a plurality of HA subtypes to their fusion- active form, and components of a lentivirus or retrovirus, wherein the recombinant pseudotyped lentiviral or retroviral vector displays HA and NA.

2. The recombinant pseudotyped lentiviral or retroviral vector of Claim 1. wherein the HA does not contain polybasic amino acids at the HA cleavage site.

3. The recombinant pseudotyped lentiviral or retroviral vector of Claim 1 or 2 wherein the protease is TMPRSS2, a trypsin protease, or a human airway trypsin-like serine protease.

4. The recombinant pseudotyped lentiviral or retroviral vector of any of the above Claims, wherein the protease is human TMPRSS2.

5. The recombinant pseudotyped lentiviral or retroviral vecto r of any of the above Claims wherein the HA is influenza HA.

6. The recombinant pseudotyped lentiviral or retroviral vecto r of any of the above Claims wherein the NA is influenza NA.

7. The recombinant pseudotyped lentiviral or retroviral vecto r of any of the above Claims wherein the HA is non-H5 influenza HA.

8. The recombinant pseudotyped lentiviral or retroviral vecto r of any of the above Claims wherein the HA is Hl, H3, H5, H7, or H9.

9. The recombinant pseudotyped lentiviral or retroviral vecto r of any of the above Claims wherein the NA is Nl , N2, N3, N7 or N8.

10. The recombinant pseudotyped lentiviral or retroviral vecto r of any of the above Claims wherein the recombinant pseudotyped lentiviral or retroviral vector further comprises a nucleic acid encoding a reporter.

11. The recombinant pseudotyped lentiviral or retroviral vecto r of any of the above Claims, wherein the reporter is a luciferase reporter.

12. The recombinant pseudotyped lentiviral or retroviral vector of any of the above Claims, which is a recombinant pseudotyped lentiviral vector.

13. A method for detecting the effectiveness of a vaccine mediated immune response comprising providing a cell in the presence of the recombinant pseudotyped lentiviral or retroviral vector of any of the above Claims and a sample from a vaccinated subject; and detecting neutralization of the recombinant pseudotyped lentiviral or retroviral vector by an antibody, if present, in the sample.

14. The method of Claim 13, wherein the antibody is labeled.

15. The method of Claim 13 or 14, wherein the detecting step further comprises detecting an amount of neutralization wherein a relatively higher amount of neutralization is indicative of a relatively more effective vaccine or greater immune response.

16. A method for detecting the potency of neutralizing monoclonal antibodies comprising providing a sample containing monoclonal antibodies in the presence of a cell and the recombinant pseudotyped lentiviral or retroviral vector of any of Claims 1-12; and detecting the ability of the monoclonal antibody to prevent entry of the recombinant pseudotyped lentiviral or retroviral vector into the cell.

17. The method of any of Claims 13-16, wherein the detecting step comprises detecting the presence of a luciferase reporter inside the cell.

18. A method of detecting a compound that affects HA-mediated viral entry into a cell comprising providing a cell in the presence of a test sample and the recombinant pseudotyped lentiviral or retroviral vector of Claim 12; and detecting recombinant pseudotyped lentiviral or retroviral vector that enters the cell.

19. The method of Claim 18, wherein the test sample is an antibody or antisera.

20. A method for detecting receptor specificity to the recombinant pseudotyped lentiviral or retroviral vector of Claim 12 comprising contacting a test cell with the recombinant pseudotyped lentiviral or retroviral vector; and detecting whether the recombinant pseudotyped lentiviral or retroviral vector binds with a cell receptor on the test cell; wherein detectable binding indicates the vector has specificity to the test cell.

21. A method for detecting an influenza infection in a subject suspected of being infected with influenza virus comprising contacting the recombinant pseudotyped lentiviral or retroviral vector of Claim 12 with a sample from the subject; and detecting binding of the recombinant pseudotyped lentiviral or retroviral vector with an antibody, if present, in the sample.

22. The method of Claim 21, wherein detectable binding indicates a protective immune response has been raised in the subject.

23. The method of any of Claims 13-15 and 21-22, wherein the sample from the vaccinated subject or the sample from the subject suspected of being infected with influenza virus is antiserum or serum.

24. The method of any of Claims 13-23 wherein the detecting is quantitative.

25. The method of any of Claims 13-24 further comprising subjecting a second sample, test cell, or test compound to the method; and comparing the results of the first and the second sample, test cell, or test compound.

26. A recombinant pseudotyped lentiviral or retroviral vector comprising a nucleic acid encoding HA, components of a lentivirus or retrovirus, wherein the vector does not comprise a nucleic acid encoding a protease.

27. The recombinant pseudotyped lentiviral or retroviral vector of Claim 26 further comprising a nucleic acid encoding NA.

28. The recombinant pseudotyped lentiviral or retroviral vector of Claim 26 or 27 wherein the HA is H5 or H7.

29. The recombinant pseudotyped lentiviral or retroviral vector of Claim 27 or 28, wherein the NA is Nl or N7.

30. A method comprising treating a pseudotyped recombinant lentiviral or retroviral vector of any of Claims 26- 29 with a protease that cleaves HA to its fusion-active form, wherein HA is expressed on the recombinant pseudotyped lentiviral or retroviral vector.

31. A kit comprising reagents used in any of the methods of Claims 13-25 and 30, the recombinant pseudotyped lentiviral or retroviral vector, and instructions for use.

32. A method of making the recombinant lentiviral or retroviral vector of any of Claims 1-12 or 26-29 comprising co-transfecting cells with the nucleic acid that encodes the HA, the nucleic acid that encodes the NA if present, and the nucleic acid that encodes the protease if present, and component of lentivirus or retrovirus under suitable transfection conditions to produce the expression vector.

33. The method of Claim 32, wherein the components of lentivirus or retrovirus further comprise a nucleic acid for encoding a reporter.

34. The method of Claim 33, wherein the components of lentivirus or retrovirus comprise a pCMVΔR8.2 plasmid and a pHR'CMV-Luc plasmid.