Patent Grant
8591916
This invention relates to viral vectors and methods employing these vectors.
Vaccination is one of the greatest achievements of medicine, and has spared millions of people the effects of devastating diseases. Before vaccines became widely used, infectious diseases killed thousands of children and adults each year in the United States alone, and so many more worldwide. Vaccination is widely used to prevent and treat infection by bacteria, viruses, and other pathogens, and also is an approach that is used in the prevention and treatment of cancer. Several different approaches are used in vaccination, including the administration of live-attenuated pathogen, killed pathogen, and inactive pathogen subunits. In the case of viral infection, live vaccines have been found to confer the most potent and durable protective immune responses.
Live-attenuated vaccines have been developed against flaviviruses, which are small, enveloped, positive-strand RNA viruses that are generally transmitted by infected mosquitoes and ticks. The Flavivirus genus of the Flaviviridae family includes approximately 70 viruses, many of which, such as yellow fever (YF), dengue (DEN), Japanese encephalitis (JE), and tick-borne encephalitis (TBE) viruses, are major human pathogens (rev. in Burke and Monath, Fields Virology, 4th Ed., p. 1043-1126, 2001).
Different approaches have been used in the development of vaccines against flaviviruses. In the case of yellow fever virus, for example, two vaccines (yellow fever 17D and the French neurotropic vaccine) were developed by serial passage (Monath, “Yellow Fever,” In Plotkin and Orenstein, Vaccines, 3rd ed., Saunders, Philadelphia, pp. 815-879, 1999). Another approach to attenuation of flaviviruses for use in vaccination involves the construction of chimeric flaviviruses, which include components of two (or more) different flaviviruses. Understanding how such chimeras are constructed requires an explanation of flavivirus structure.
Flavivirus proteins are produced by translation of a single, long open reading frame to generate a polyprotein, which is followed by a complex series of post-translational proteolytic cleavages of the polyprotein by a combination of host and viral proteases to generate mature viral proteins (Amberg et al., J. Virol. 73:8083-8094, 1999; Rice, “Flaviviridae,” In Virology, Fields (ed.), Raven-Lippincott, New York, 1995, Volume I, p. 937). The virus structural proteins are arranged in the polyprotein in the order C-prM-E, where “C” is capsid, “prM” is a precursor of the viral envelope-bound M protein, and “E” is the envelope protein. These proteins are present in the N-terminal region of the polyprotein, while the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are located in the C-terminal region of the polyprotein.
Chimeric flaviviruses have been made that include structural and non-structural proteins from different flaviviruses. For example, the so-called CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) technology employs the yellow fever 17D virus capsid and nonstructural proteins to deliver the envelope proteins (M and E) of other flaviviruses (see, e.g., Chambers et al., J. Virol. 73:3095-3101, 1999). This technology has been used to develop vaccine candidates against dengue, Japanese encephalitis (JE), West Nile (WN), and St. Louis encephalitis (SLE) viruses (see, e.g., Pugachev et al., in New Generation Vaccines, 3rd ed., Levine et al., eds., Marcel Dekker, New York, Basel, pp. 559-571, 2004; Chambers et al., J. Virol. 73:3095-3101, 1999; Guirakhoo et al., Virology 257:363-372, 1999; Monath et al., Vaccine 17:1869-1882, 1999; Guirakhoo et al., J. Virol. 74:5477-5485, 2000; Arroyo et al., Trends Mol. Med. 7:350-354, 2001; Guirakhoo et al., J. Virol. 78:4761-4775, 2004; Guirakhoo et al., J. Virol. 78:9998-10008, 2004; Monath et al., J. Infect. Dis. 188:1213-1230, 2003; Arroyo et al., J. Virol. 78:12497-12507, 2004; and Pugachev et al., Am. J. Trop. Med. Hyg. 71:639-645, 2004).
CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-based vaccines have been shown to have favorable properties with respect to properties such as replication in substrate cells, low neurovirulence in murine models, high attenuation in monkey models, high genetic and phenotypic stability in vitro and in vivo, inefficient replication in mosquitoes (which is important to prevent uncontrolled spread in nature), and the induction of robust protective immunity in mice, monkeys, and humans following administration of a single dose, without serious post-immunization side effects. Indeed, the CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-JE vaccine virus, containing the prM-E genes from the SA14-14-2 JE virus (live attenuated JE vaccine used in China), was successfully tested in preclinical and Phase I and II clinical trials (Monath et al., Vaccine 20:1004-1018, 2002; Monath et al., J. Infect. Dis. 188:1213-1230, 2003). Similarly, successful Phase I clinical trials have been conducted with a CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-WN vaccine candidate, which contains prM-E sequences from a West Nile virus (NY99 strain), with three specific amino acid changes incorporated into the E protein to increase attenuation (Arroyo et al., J. Virol. 78:12497-12507, 2004).
In addition to being used as vaccines against flavivirus infection, flaviviruses, such as chimeric flaviviruses, have been proposed for use as vectors for the delivery of other, non-flavivirus peptides. In one example of such a use, a rational approach for insertion of foreign peptides into the envelope protein of YF17D virus was described, based on knowledge of the tertiary structure of the flavivirus particle, as resolved by cryoelectron microscopy and fitting the known X-ray structure of the protein dimer into an electron density map (Rey et al., Nature 375:291-298, 1995; Kuhn et al., Cell 108:717-725, 2002). The three-dimensional structure of the protein trimer in its post-fusion conformation has also been resolved (Modis et al., Nature 427:313-319, 2004; Bressanelli et al., EMBO J. 23:728-738, 2004). Galler and co-workers examined the three-dimensional structures of the envelope protein dimer and trimer and concluded that the fg loop of dimerization domain II should be solvent-exposed in both the dimer and trimer conformations. They used this loop to insert malaria humoral and T-cell epitopes into the envelope protein of YF17D virus and recovered a few viable mutants (Bonaldo et al., J. Virol. 79:8602-8613, 2005; Bonaldo et al., J. Mol. Biol. 315:873-885, 2002; WO 02/072835). Use of this approach, however, does not ensure that a selected site is permissive/optimal for the insertion of every desired foreign peptide in terms of efficient virus replication (as evidenced by some of the Galler et al. data), immunogenicity, and stability. Further, this approach is not applicable to viral proteins for which three-dimensional structures are unknown (e.g., prM/M, NS1, and most other NS proteins of flaviviruses).
In another approach, the envelope protein of CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-JE was probed for permissive insertion sites using a transposon. According to this approach, an inserted transposon in a viable mutant virus is replaced with a desired foreign peptide (see, e.g., WO 02/102828). In yet another approach, foreign sequences were inserted into the yellow fever virus strain YF-17D, downstream of the polyprotein open reading frame (US 2004/0241821).
The invention provides flaviviruses that include one or more insertions of sequences encoding a heterologous peptide or protein between (i) nucleotides encoding amino acids corresponding to amino acids 277 and 278 of the envelope protein of Japanese encephalitis virus, (ii) nucleotides encoding amino acids corresponding to amino acids 207 and 208 of the envelope protein of Japanese encephalitis virus, or (iii) nucleotides encoding amino acids within five amino acids of those corresponding to amino acids 277 and 278, or amino acids 207 and 208, of the envelope protein of Japanese encephalitis virus.
By amino acids “corresponding to” the indicated Japanese encephalitis amino acids is meant, in addition to the indicated amino acids of Japanese encephalitis virus, amino acids in envelope proteins of other flaviviruses that align with these or closely positioned amino acids, as can readily be determined by those of skill in the art (see, e.g., below and
The flaviviruses of the invention can be chimeric flaviviruses, including structural proteins of a first flavivirus and non-structural proteins of a second flavivirus (e.g., a yellow fever virus, such as YF17D (also see below). For example, the flaviviruses can include pre-membrane and envelope proteins of the first flavivirus and capsid and non-structural proteins of the second flavivirus.
Examples of first flaviviruses that can be included in the chimeric flaviviruses of the invention include Japanese encephalitis, Dengue-1, Dengue-2, Dengue-3, Dengue-4, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Ilheus, Tick-borne encephalitis, Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses.
The heterologous peptides or proteins encoded by heterologous sequences of the flaviviruses of the invention can be, e.g., vaccine antigens. Such vaccine antigens can be derived from an infectious agent, such as an influenza virus. Examples of such vaccine antigens include hemagglutinin, neuraminidase, M2, and immunogenic fragments thereof (e.g., the M2e region of the M2 protein or a fragment thereof, such as peptides of the sequences MSLLTEVETPIR (SEQ ID NO:1) or MSLLTEVETPIRNEWGSRSNDSSD (SEQ ID NO:2)), which may also include amino and/or carboxy terminal glycine linker sequences (e.g., 1 or 2 glycines on either or both ends). In other examples, the heterologous peptide or protein is present between nucleotides encoding amino acids corresponding to amino acids 277 and 278 of the envelope protein of Japanese encephalitis virus, and/or the heterologous peptide or protein is present between nucleotides encoding amino acids corresponding to amino acids 207 and 208 of the envelope protein of Japanese encephalitis virus. Specific examples of inserted sequences that can be used in the invention include those selected from the group consisting of: SEQ ID NOs:1, 2, 13-15, 20-59, and 65-76.
Further, the flaviviruses of the invention may optionally include a deletion of 3′-untranslated region and/or the NS1 sequences, as described further below.
The invention also includes methods of administering protein and/or peptides to subjects, which involve administration of the flaviviruses described above or elsewhere herein.
Also featured in the invention are nucleic acid molecules encoding the flaviviruses described above or elsewhere herein, as well as pharmaceutical compositions including such flaviviruses.
The invention also includes methods of producing flaviviruses such as those described above and elsewhere herein, which involve culturing cells into which RNA corresponding to the viruses has been introduced at a temperature below 37° C. (e.g., 30° C.-36° C. or 34° C.). Further, the invention includes methods of propagating the flaviviruses, which involve incubating cells infected with the viruses at a temperature below 37° C. (e.g., 30° C.-36° C. or 34° C.).
Also included in the invention are flavivirus replicons including one or more insertions of sequences encoding a heterologous peptide or protein between (i) nucleotides encoding amino acids corresponding to amino acids 277 and 278 of the envelope protein of Japanese encephalitis virus, (ii) nucleotides encoding amino acids corresponding to amino acids 207 and 208 of the envelope protein of Japanese encephalitis virus, or (iii) nucleotides encoding amino acids within five amino acids of those corresponding to amino acids 277 and 278, or amino acids 207 and 208, of the envelope protein of Japanese encephalitis virus. Corresponding pharmaceutical compositions, as well as therapeutic and prophylactic methods, are also included in the invention.
The invention provides several advantages. For example, the live, attenuated viral vectors of the invention induce strong, long-lasting immune responses against specific antigens. The vectors of the invention can be used to confer immunity to infectious diseases, such as influenza, or to disease-related proteins such as cancer antigens and the like. As an example, the invention can be used to deliver influenza virus M2e (or a fragment thereof), which is the external portion of M2, a minor influenza A surface protein that is conserved among diverse influenza viruses and may serve as the basis for a vaccine that protects against all influenza A strains (Neirynck et al., Nat. Med. 5(10):1157-1163, 1999; Fiers et al., Virus Res. 103(1-2):173-176, 2004).
An additional advantage of the vectors of the invention is that, as described further below, they can be used to deliver relatively large antigens, as compared to many previously known viral vectors. Thus, as an example, in addition to M2e, the vectors of the invention can advantageously be used to administer larger portions of M2 or even full length M2.
The advantages of using live vectors, such as the flavivirus-based vectors of the invention, also include (i) expansion of the antigenic mass following vaccine inoculation; (ii) the lack of need for an adjuvant; (iii) the intense stimulation of innate and adaptive immune responses (YF17D, for example, is the most powerful known immunogen); (iv) the possibility of more favorable antigen presentation due to, e.g., the ability of CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) (derived from YF17D) to infect antigen presenting cells, such as dendritic cells and macrophages; (v) the possibility to obtain a single-dose vaccine providing life-long immunity; (vi) the envelopes of CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) vaccine viruses are easily exchangeable, giving a choice of different recombinant vaccines, some of which are more appropriate than the others in different geographic areas or for sequential use; (vii) the possibility of modifying complete live flavivirus vectors into packaged, single-round-replication replicons, in order to eliminate the chance of adverse events or to minimize the effect of anti-vector immunity during sequential use; and (viii) the low cost of manufacture.
Additional advantages provided by the invention relate to the fact that chimeric flavivirus vectors of the invention are sufficiently attenuated so as to be safe, and yet are able to induce protective immunity to the flaviviruses from which the proteins in the chimeras are derived and, in particular, the proteins or peptides inserted into the chimeras. Additional safety comes from the fact that some of the vectors used in the invention are chimeric, thus eliminating the possibility of reversion to wild type. An additional advantage of the vectors of the invention is that flaviviruses replicate in the cytoplasm of cells, so that the virus replication strategy does not involve integration of the viral genome into the host cell, providing an important safety measure. Further, a single vector of the invention can be used to deliver multiple epitopes from a single antigen, or epitopes derived from more than one antigen.
An additional advantage provided by the invention relates to the use of new growth conditions for propagating viral vectors, such as those described herein. As is discussed further below, these conditions enable the production of relatively high titer virus, with increased immunogenicity.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.
The invention provides live, attenuated viral vectors that can be used in the administration of vaccine antigens, such as vaccine antigens against influenza virus. Also included in the invention are methods of using these vectors in methods for preventing and treating influenza virus infection, pharmaceutical compositions including the vectors, and nucleic acid molecules corresponding to genomes of the viral vectors or the complements thereof. As discussed further below, the viral vaccine vectors of the invention can be used to induce long-lasting immune responses against specific influenza antigens. For example, the vaccine vectors of the present invention can be used to express a universal influenza antigen that is inserted into a highly immunogenic site, the flavivirus envelope (E) protein. The invention provides compositions intended to protect animals, including humans, against a broad range of influenza strains. Further, the invention provides methods of making and propagating viral vectors such as those of the invention. The vectors, methods, and compositions of the invention are described further, as follows.
Viral Vectors
In certain examples, the vectors of the invention are based on CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) viruses, which, as described above, consist of a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus. For example, the chimeras can consist of a first flavivirus in which the prM and E proteins have been replaced with the prM and E proteins of a second flavivirus. As is discussed above, flavivirus proteins, including those of the chimeric flaviviruses described herein, are produced as a polyprotein that is post-translationally cleaved into subunit proteins: the amino terminal structural proteins, capsid (C), pre-membrane (prM), and envelope (E), and the carboxyl terminal non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.
As is described further below, the vectors of the invention include insertions of influenza virus sequences, such as influenza virus M2e sequences. Three specific examples of such vectors, which are based on chimeric yellow fever/Japanese encephalitis viruses, are described below. In these vectors, influenza sequences, such as M2e sequences, are inserted between Japanese encephalitis virus envelope amino acids 277 and 278 (ES275:M2e and E277M2e viruses) or (ii) between amino acids 207 and 208 sequence (EG202:M2e virus). The inserts in these examples have the following amino acid sequences: (i) (G)1-2MSLLTEVETPIRGG (SEQ ID NOs:13 or 15), comprising an N-terminal one- or two-glycine linker, followed by the first 12 amino acids of influenza protein M2, followed in turn by a C-terminal two-glycine linker, and (i) GGMSLLTEVETPIRNEWGSRSNDSSDGG (SEQ ID NO:14), comprising first 24 amino acids of influenza M2 protein flanked from both terminus by two-glycine linkers. Additional details concerning these examples are provided below.
In addition to positions corresponding to those of Japanese encephalitis virus between amino acids 277 and 278, and amino acids 207 and 208, the invention also includes vectors in which inserts are made at different sites in these areas. Thus, for example, the vectors can include insertions between the following pairs of amino acids: 272/273, 273/274, 274/275, 275/276, 276/277, 278/279, 279/280, 280/281, 281/282, 282/283; 202/203, 203/204, 204/205, 205/206, 206/207, 208/209, 209/210, 210/211, 211/212, and 212/213.
In the case of non-JE sequences, the insertions can be made, for example, between amino acids 203 and 204 or amino acids 280 and 281 of Tick-borne encephalitis virus; between amino acids 199 and 200 or amino acids 273 and 274 of yellow fever virus; between amino acids 207 and 208 or amino acids 278 and 279 of West Nile virus; and between amino acids 202 and 203 or amino acids 275 and 276 of Dengue-4 virus. Similar to JE, as discussed above, the invention includes vectors in which insertions are made in non-JE sequences within 5 amino acids of the sites corresponding to the JE insertion sites, as noted above.
The chimeric viruses that are used in the invention can be made from any combination of flaviviruses. As is noted above, the chimeras can include structural proteins from a first flavivirus (pre-membrane (prM), envelope (E), and/or capsid (C)) and non-structural proteins from a second, different flavivirus (or flavivirus serotype). For example, the chimeras can include pre-membrane and envelope proteins from a first flavivirus and capsid and non-structural proteins from a second flavivirus.
Specific examples of chimeras that can be used in the invention include yellow fever virus capsid and non-structural sequences, and Japanese encephalitis virus pre-membrane and envelope sequences. However, other viruses can be used as well. Examples of particular flaviviruses that can be used in the invention, as first or second viruses, include mosquito-borne flaviviruses, such as Japanese encephalitis, Dengue (serotypes 1-4), yellow fever, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, and Ilheus viruses; tick-borne flaviviruses, such as Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses; as well as viruses from the Hepacivirus genus (e.g., Hepatitis C virus).
Details of making chimeric viruses that can be used in the invention are provided, for example, in U.S. Pat. Nos. 6,962,708 and 6,696,281; PCT international applications WO 98/37911 and WO 01/39802; and Chambers et al., J. Virol. 73:3095-3101, 1999, the contents of each of which are incorporated by reference herein in its entirety. In addition, these chimeric viruses can include attenuating mutations, such as those described in the following documents, the contents of each of which is incorporated herein by reference: WO 2003/103571; WO 2005/082020; WO 2004/045529; WO 2006/044857; PCT/US2006/015241; U.S. Pat. No. 6,685,948 B1; U.S. Patent Application Publication US 2004/0052818 A1; U.S. Patent Application Publication 2005/0010043 A1; WO 02/074963; WO 02/095075 A1; WO 03/059384 A1; WO 03/092592 A2; as well as the documents cited above.
A specific example of a type of chimeric virus that can be used in the invention is the human yellow fever virus vaccine strain, YF17D, in which the prM and E proteins have been replaced with prM and E proteins of another flavivirus, such as Japanese encephalitis virus, West Nile virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, a Dengue virus, or any other flavivirus, such as one of those listed above. For example, the following chimeric flaviviruses, which were deposited with the American Type Culture Collection (ATCC) in Manassas, Va., U.S.A. under the terms of the Budapest Treaty and granted a deposit date of Jan. 6, 1998, can be used in the invention: Chimeric Yellow Fever 17D/Japanese Encephalitis SA14-14-2 Virus (YF/JE A1.3; ATCC accession number ATCC VR-2594) and Chimeric Yellow Fever 17D/Dengue Type 2 Virus (YF/DEN-2; ATCC accession number ATCC VR-2593).
Among the advantages of using the CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) vaccines as vectors, a main advantage is that the envelope proteins (which are the main antigenic determinants of immunity against flaviviruses, and in this case, anti-vector immunity) can be easily exchanged allowing for the construction of several different vaccines using the same YF17D backbone that can be applied sequentially to the same individual. In addition, different recombinant CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) insertion vaccines can be determined to be more appropriate for use in specific geographical regions in which different flaviviruses are endemic, as dual vaccines against an endemic flavivirus and another targeted pathogen. For example, CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-JE-influenza vaccine may be more appropriate in Asia, where JE is endemic, to protect from both JE and influenza, YF17D-influenza vaccine may be more appropriate in Africa and South America, where YF is endemic, CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-WN-influenza may be more appropriate for the U.S. and parts of Europe and the Middle East, in which WN virus is endemic, and CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-Dengue-influenza-may be more appropriate throughout the tropics where dengue viruses are present.
In addition to chimeric flaviviruses, other flaviviruses, such as non-chimeric flaviviruses, can be used as vectors according to the present invention. Examples of such viruses that can be used in the invention include live, attenuated vaccines, such as those derived from the YF17D strain, which was originally obtained by attenuation of the wild-type Asibi strain (Smithburn et al., “Yellow Fever Vaccination,” World Health Organization, p. 238, 1956; Freestone, in Plotkin et al. (eds.), Vaccines, 2nd edition, W.B. Saunders, Philadelphia, U.S.A., 1995). An example of a YF17D strain from which viruses that can be used in the invention can be derived is YF17D-204 (YF-VAX®, Sanofi-Pasteur, Swiftwater, Pa., USA; Stamaril®, Sanofi-Pasteur, Marcy-L'Etoile, France; ARILVAX™, Chiron, Speke, Liverpool, UK; FLAVIMUN®, Berna Biotech, Bern, Switzerland; YF17D-204 France (X15067, X15062); YF17D-204, 234 US (Rice et al., Science 229:726-733, 1985)), while other examples of such strains that can be used are the closely related YF17DD strain (GenBank Accession No. U 17066), YF17D-213 (GenBank Accession No. U17067), and yellow fever virus 17DD strains described by Caller et al., Vaccines 16(9/10):1024-1028, 1998. In addition to these strains, any other yellow fever virus vaccine strains found to be acceptably attenuated in humans, such as human patients, can be used in the invention.
Further, in addition to live viruses, as discussed above, packaged replicons expressing foreign proteins or peptides can be used in the invention. This approach can be used, for example, in cases in which it may be desirable to increase safety or to minimize antivector immunity (neutralizing antibody response against the envelope proteins), in order to use the same vector for making different vaccines that can be applied to the same individual. Technology for the construction of single-round replicons is well established, and the immunogenic potential of replicons has been demonstrated (Jones et al., Virology 331:247-259, 2005; Molenkamp et al., J. Virol. 77:1644-1648, 2003; Westaway et al., Adv. Virus. Res. 59:99-140, 2003). In an example of such a replicon, most of the prM and E envelope protein genes are deleted. Therefore, it can replicate inside cells, but cannot generate virus progeny (hence single-round replication). It can be packaged into viral particles when the prM-E genes are provided in trans. Still, when cells are infected by such packaged replicon (e.g., following vaccination), a single round of replication follows, without further spread to surrounding cell/tissues.
Protective epitopes from different pathogens can be combined in one virus, resulting in triple-, quadruple-, etc., vaccines. Also, a CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or Proteins) of a second virus) variant containing the envelope from a non-endemic flavivirus can be used to avoid the risk of natural antivector immunity in a population that otherwise could limit the effectiveness of vaccination in a certain geographical area (e.g., CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-JE vector may be used in the U.S. where JE is not present).
Heterologous Proteins and Peptides
The vectors of the invention can be used to deliver or produce any peptide or protein of prophylactic, therapeutic, diagnostic, or experimental interest. For example, the vectors can be used in the induction of an immune response (prophylactic or therapeutic) against any protein-based antigen that is inserted in the envelope protein (e.g., between amino acids 277 and 278 of the E protein, or between amino acids 207 and 208, as described above and elsewhere herein. In some cases, it may be desirable to maintain the size of the flavivirus into which an insert gene is introduced, as much as possible, in order to maintain virus genetic stability and viability. This can be achieved, for example, by the deletion of sequences in the 3′-untranslated region of the virus (see below and also U.S. Pat. No. 6,685,948; US 2005/0010043 A1; PCT/US2006/015241; WO 02/074963; WO 02/095075 A1; WO 03/059384 A1; and WO 03/092592 A2; also see Deubel et al., “Biological and Molecular Variations of Yellow Fever Virus Strains,” In Saluzzo et al. (eds.), “Factors in the Emergence of Arbovirus Diseases” Elsevier, Paris, 1997, pages 157-165).
In another example, portions of the NS1 gene (e.g., all or most of the NS1 gene) can be deleted to accommodate an insert. The elimination of NS1 (ΔNS1), which is about 1.2 kb in length, allows the insertion of transgenes similar in size. A consequence of this deletion is that the NS1 function must now be supplied in trans by introduction of the NS1 gene into the cell line used to produce a ΔNS 1 chimera (see, e.g., Lindenbach et al., J. Virol. 71:9608-9617, 1997). The chimeric viral particles produced in this way can infect cells, but are not capable of replication in vivo. This creates an antigen gene-delivery vector, which, in addition to avoiding potential problems with genome size limitations, has different properties from the replication-competent chimeras described above (e.g., decreased virulence).
Antigens that can be used in the invention can be derived from, for example, infectious agents such as viruses, bacteria, and parasites. A specific example of such an infectious agent is influenza viruses, including those that infect humans (e.g., A (e.g., strain A/HK/8/68), B, and C strains), as well as avian influenza viruses. A specific example of an epitope that can be included in the vectors of the invention is the M2e epitope of influenza A (strain A/HK/8/68). One example of such an epitope consists of an insert has the following amino acid sequence: (G)1-2MSLLTEVETPIRGG (SEQ ID NOs:13 or 15), comprising an N-terminal one- or two-glycine linker, followed by the first 12 amino acids of influenza protein M2, followed in turn by a C-terminal two-glycine linker.
Other examples of antigens from influenza viruses include those derived from hemagglutinin (HA; e.g., any one of H1-H16, or subunits thereof) (or HA subunits HA1 and HA2), neuraminidase (NA; e.g., any one of N1-N9), M2 (e.g., M2e), M1, nucleoprotein (NP), and B proteins. For example, peptides including the hemagglutinin precursor protein cleavage site (HA0) (e.g., NIPSIQSRGLFGAIAGFIE (SEQ ID NO:20) for A/H1 strains, NVPEKQTRGIFGAIAGFIE (SEQ ID NO:21) for A/H3 strains, and PAKLLKERGFFGAIAGFLE (SEQ ID NO:22) for influenza B strains), or HA peptide SKAFSNCYPYDVPDYASL (SEQ ID NO:23), or its variant SKAFSNSYPYDVPDYASL (SEQ ID NO:24), or M2e (e.g., MSLLTEVETPIRNEWGSRSNDSSD (SEQ ID NO:2); also see European Patent No. 0 996 717 B1, the contents of which are incorporated herein by reference), as well as peptide sequences listed in supplementary table 10 of Bui et al., Proc. Natl. Acad. Sci. U.S.A. 104:246-251, 2007, can be used (SEQ ID NOs:65-76). Other examples of peptides that are conserved in influenza can be used in the invention and include: NBe peptide conserved for influenza B (e.g., consensus sequence MNNATFNYTNVNPISHIRGS (SEQ ID NO:25)); the extracellular domain of BM2 protein of influenza B (e.g., consensus MLEPFQ (SEQ ID NO:26)); and the M2e peptide from the H5N1 avian flu (e.g., MSLLTEVETLTRNGWGCRCSDSSD (SEQ ID NO:27)). Use of influenza virus M2 (or fragments thereof, such as M2e) is particularly advantageous, because the sequence of this protein is highly conserved, as compared with the sequences of other influenza proteins (see, e.g., European Patent 0 996 717 B1).
Further examples of influenza proteins and peptides that can be used in the invention, as well as proteins from which such peptides can be derived (e.g., by fragmentation) are described in US 2002/0165176, US 2003/0175290, US 2004/0055024, US 2004/0116664, US 2004/0219170, US 2004/0223976, US 2005/0042229, US 2005/0003349, US 2005/0009008, US 2005/0186621, U.S. Pat. No. 4,752,473, U.S. Pat. No. 5,374,717, U.S. Pat. No. 6,169,175, U.S. Pat. No. 6,720,409, U.S. Pat. No. 6,750,325, U.S. Pat. No. 6,872,395, WO 93/15763, WO 94/06468, WO 94/17826, WO 96/10631, WO 99/07839, WO 99/58658, WO 02/14478, WO 2003/102165, WO 2004/053091, WO 2005/055957, and Tables 1-4 (and references cited therein), the contents of which are incorporated by reference.
Protective epitopes from other human/veterinary pathogens, such as parasites (e.g., malaria), other pathogenic viruses (e.g., human papilloma virus (HPV), herpes simplex viruses (HSV), human immunodeficiency viruses (HIV), and hepatitis C viruses (HCV)), and bacteria (e.g., Mycobacterium tuberculosis, Clostridium difficile, and Helicobacter pylori) can also be included in the vectors of the invention. Examples of additional pathogens, as well as antigens and epitopes from these pathogens, which can be used in the invention are provided in WO 2004/053091, WO 03/102165, WO 02/14478, and US 2003/0185854, the contents of which are incorporated herein by reference. Further, additional therapeutic protein/antigen sources that can be included in the vectors of the present invention are listed in US 2004/0241821, which is incorporated herein by reference.
Additional examples of pathogens from which antigens can be obtained are listed in Table 5, below, and specific examples of such antigens include those listed in Table 6. In addition, specific examples of epitopes that can be inserted into the vectors of the invention are provided in Table 7. As is noted in Table 7, epitopes that are used in the vectors of the invention can be B cell epitopes (i.e., neutralizing epitopes) or T cell epitopes (i.e., T helper and cytotoxic T cell-specific epitopes).
The vectors of the invention can be used to deliver antigens in addition to pathogen-derived antigens. For example, the vectors can be used to deliver tumor-associated antigens for use in immunotherapeutic methods against cancer. Numerous tumor-associated antigens are known in the art and can be administered according to the invention. Examples of cancers (and corresponding tumor associated antigens) are as follows: melanoma (NY-ESO-1 protein (specifically CTL epitope located at amino acid positions 157-165), CAMEL, MART 1, gp100, tyrosine-related proteins TRP1 and 2, and MUC1)); adenocarcinoma (ErbB2 protein); colorectal cancer (17-1A, 791Tgp72, and carcinoembryonic antigen); prostate cancer (PSA1 and PSA3). Heat shock protein (hsp110) can also be used as such an antigen. (Also see, e.g., US 2004/0241821 for additional examples.)
In another example of the invention, exogenous proteins that encode an epitope(s) of an allergy-inducing antigen to which an immune response is desired can be used.
The size of the protein or peptide that is inserted into the vectors of the invention can range in length from, for example, from 5-1500 amino acids in length, for example; from 8-1000, 10-500, 10-100, 10-50, 10-35, or 12-20 amino acids in length, as can be determined to be appropriate by those of skill in the art. In addition, the proteins or peptides noted herein can include additional sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) or can be reduced in length, also as can be determined to be appropriate by those skilled in the art. Further, as is described elsewhere herein, deletions can be made in the vectors of the invention to accommodate different sized inserts, as determined to be appropriate by those of skill in the art.
Production and Administration
The viruses described above can be made using standard methods in the art. For example, an RNA molecule corresponding to the genome of a virus can be introduced into primary cells, chicken embryos, or diploid cell lines, from which (or from the supernatants of which) progeny virus can then be purified. Other methods that can be used to produce the viruses employ heteroploid cells, such as Vero cells (Yasumura et al., Nihon Rinsho 21:1201-1215, 1963). In an example of such methods, a nucleic acid molecule (e.g., an RNA molecule) corresponding to the genome of a virus is introduced into the heteroploid cells, virus is harvested from the medium in which the cells have been cultured, harvested virus is treated with a nuclease (e.g., an endonuclease that degrades both DNA and RNA, such as BENZONASE™ (endonuclease from Serratia marcescens); U.S. Pat. No. 5,173,418), the nuclease-treated virus is concentrated (e.g., by use of ultrafiltration using a filter having a molecular weight cut-off of, e.g., 500 kDa), and the concentrated virus is formulated for the purposes of vaccination. Details of this method are provided in WO 03/060088 A2, which is incorporated herein by reference. Further, methods for producing chimeric viruses are described in the documents cited above in reference to the construction of chimeric virus constructs.
The vectors of the invention are administered to subjects (e.g., humans and non-human animals, such as horses, livestock, and domestic pets (e.g., cats and dogs)) in amounts and by using methods that can readily be selected by persons of ordinary skill in this art. In the case of chimeric flaviviruses and yellow fever virus-based vectors, the vectors can be administered and formulated, for example, in the same manner as the yellow fever 17D vaccine, e.g., as a clarified suspension of infected chicken embryo tissue, or a fluid harvested from cell cultures infected with the chimeric yellow fever virus. The vectors of the invention can thus be formulated as sterile aqueous solutions containing between 100 and 1,000,000 infectious units (e.g., plaque-forming units or tissue culture infectious doses) in a dose volume of 0.1 to 1.0 ml, to be administered by, for example, intramuscular, subcutaneous, or intradermal routes (see, e.g., WO 2004/0120964 for details concerning intradermal vaccination approaches). In addition, because flaviviruses may be capable of infecting the human host via the mucosal routes, such as the oral route (Gresikova et al., “Tick-borne Encephalitis,” In The Arboviruses, Ecology and Epidemiology, Monath (ed.), CRC Press, Boca Raton, Fla., 1988, Volume IV, 177-203), the vectors can be administered by a mucosal route. The vectors of the invention can be administered in “effective amounts,” which are amounts sufficient to produce a desired effect, such as induction of an immune response (e.g., a specific immune response) and/or amelioration of one or more symptoms of a disease or condition.
When used in immunization methods, the vectors can be administered as primary prophylactic agents in adults or children (or animals; see above) at risk of infection by a particular pathogen. The vectors can also be used as secondary agents for treating infected subjects by stimulating an immune response against the pathogen from which the peptide antigen is derived. Further, an epitope, peptide, or protein is “administered” to a subject according to the methods described herein, whether it is present in the material that is actually administered, or is generated by progeny viruses that replicate from the administered material.
For vaccine applications, optionally, adjuvants that are known to those skilled in the art can be used. Adjuvants that can be used to enhance the immunogenicity of the chimeric vectors include, for example, liposomal formulations, synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine. Although these adjuvants are typically used to enhance immune responses to inactivated vaccines, they can also be used with live vaccines. In the case of a chimeric vector delivered via a mucosal route, for example, orally, mucosal adjuvants such as the heat-labile toxin of E. coli (LT) or mutant derivations of LT can be used as adjuvants. In addition, genes encoding cytokines that have adjuvant activities can be inserted into the vectors. Thus, genes encoding cytokines, such as GM-CSF, IL-2, IL-12, IL-13, or IL-5, can be inserted together with foreign antigen genes to produce a vaccine that results in enhanced immune responses, or to modulate immunity directed more specifically towards cellular, humoral, or mucosal responses. In addition to vaccine applications, as those skilled in the art can readily understand, the vectors of the invention can be used in gene therapy methods to introduce therapeutic gene products into a patient's cells and in cancer therapy.
The invention also provides methods for producing viral vectors such as those described herein, in which cells (e.g., Vero cells) transfected with RNA corresponding to the vectors are advantageously cultured at a temperature below 37° C., e.g., 30-36° C., 31-35° C., or 32-34° C. As is described further below, culturing of such transfected cells at 34° C. resulted in the production of virus at higher titers, and presumably with a corresponding increase in antigen production, since the antigen is an integral part of the viral envelope protein. Thus, the invention provides an improved method for the production of flavivirus vaccines, such as those described herein.
Experimental Results
CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) technology can be used to induce immunity against antigens that are not of flavivirus origin. This requires the insertion of these antigens in the genome of a CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus) vaccine such as CV-JE, while preserving the viability of the virus and without causing excessive genetic instability. The present invention provides a CHIMERIVAX™ (a first flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been replaced with a corresponding structural protein (or proteins) of a second virus)-JE-Influenza vaccine that is viable and appears genetically stable. While CV-JE is normally grown in tissue culture at 37° C., the use of lower incubation temperatures to propagate an engineered virus bearing an inserted antigen in the E protein is shown to improve genetic stability.
An antigen of interest in this study is the M2e epitope of influenza A (strain A/HK/8/68). More specifically, the insert can have the following amino acid sequences: (G)1-2MSLLTEVETPIRGG (SEQ ID NO:13 or 15), comprising an N-terminal one- or two-glycine linker, followed by the first 12 amino acids of influenza protein M2, followed in turn by a C-terminal two-glycine linker; and GGMSLLTEVETPIRNEWGSRSNDSSDGG (SEQ ID NO:14), comprising the first 24 amino acids of the influenza M2 protein flanked on both termini with two-glycine linkers. The cDNA encoding these peptides was inserted in the CV-JE genome in such a way that the peptide is: (i) between amino acids 277 and 278 of the E protein (ES275:M2e and E277M2e viruses), or (ii) between amino acids 207 and 208 (EG202:M2e virus). This strategy is generally illustrated in
Both insertion sites were identified by analysis of structural information of the Japanese encephalitis virus E protein model, which was based on the template of the West Nile virus envelope glycoprotein, in combination with multiple alignment comparisons of the amino acid sequence of Japanese encephalitis with those of several distant members of the Flavivirus family. Table 8 links the names of the constructs with insertion site and insert sequence.
The insertion of the (G)1-2MSLLTEVETPIRGG (SEQ ID NO:13 or 15) peptide epitope was carried out, using standard methods, by overlap PCR followed by cloning of the overlap PCR product into plasmid pBSA, which is a bacterial artificial chromosome containing the entire CV-JE genome. The resulting new DNA construct was sequenced according to standard methods to verify that the intended sequence, including the peptide insert, was present. Capped viral genomic RNA was produced by in vitro transcription using the engineered pBSA construct as a template, and this RNA was then transfected into Vero cells using lipofectamine 2000. The transfected cells were grown at 34° C. for 7 days and culture supernatants were harvested (identified as P1) and used to infect fresh culture flasks. After 5 days, these second cultures (P2) were also harvested. In
Supernatants P1 and P2 of the EG202:M2e virus were used to infect Vero cells overlaid with agarose to produce plaques. The infected cells were incubated at 34° C. for 5 days, the plaques were then visualized by adding a neutral red-containing overlay for 1 additional day, and 12 individual plaques were picked to isolate single viral clones. These plaque picks were resuspended in a small volume of growth medium and an aliquot of 250 μl was used to inoculate 6 cultures at either 34° C. or 37° C. The cultures were then incubated for 6 days and supernatants harvested. RT-PCR was carried out to determine whether insert-bearing viral isolates were present (
Insertion of GGMSLLTEVETPIRNEWGSRSNDSSDGG (SEQ ID NO:14) peptide at position 277 of Japanese encephalitis virus E protein was carried out essentially using the same methods as described for the (G)1-2MSLLTEVETPIRGG (SEQ ID NO:13 or 15) epitope. A DNA fragment derived by overlap PCR was cloned into the pBSA vector, and the presence of insert was confirmed by sequence analysis. Infectious RNA was transcribed from XhoI linearized vector with the insert using the advantage of the SP6 promoter located immediately upstream of the region encoding the full-length genome of CV-JE virus, and used to transfect Vero cells with lipofectamine 2000 reagent. Transfected Vero cells were incubated at 37° C. in the presence of 5% CO2 for 6-7 days until the first sign of cytopathic effect; then culture medium was harvested and an additional round of virus amplification was performed on fresh cells. This resulted in uncloned virus designated as E277M2e herein, which represented a heterologous virus population based on a pattern of plaques developed in Vero cells under methyl cellulose overlay, and stained using a standard immunofocus protocol (
Homologous virus was produced by two plaque purifications of E277M2e and selected virus clones were amplified twice on Vero cells to produce a viral stock, identified as P2. One of the E277M2e clones with a large plaque phenotype, designated herein as E277M2e-P2 displayed a uniform plaque morphology on Vero cells, and all foci were equally stained with Mab 14C2 (
Conformation of the M2e epitope and its presence on the surface of the virion when expressed with E protein at position 277 was assayed in a neutralization assay. Monoclonal antibody 14C2, recognizing only a continuous M2e epitope, effectively neutralized E277M2e-P2 virus, with PRNT50 titer ≧819,200 (approximately 100 pg of antibody); polyclonal antibody raised against M2e protein within a Hepatitis B core-M2e (HBc-M2e) particle (Acam-FluA vaccine had a similar effect on E277M2e-P2 neutralization, with PRNT50 titer of ≧81,920 (
To test the immunogenicity of the M2e epitope delivered by CVJE live virus vector, 4 week old female Balb/C mice in groups of 8-10 animals were intraperitoneally immunized and boosted 30 days after prime with 6 log10 PFU of E77M2e-P2 virus. Two control groups were treated similarly with either 7 log10 PFU/ml of CVJE parental virus or 10 μg of Acam-FluA adjuvanted with aluminum hydroxide. On day 60, after the boosting dose, all animals were bled and humoral immune response was assayed in end-point ELISA against M2e synthetic peptide (
Protectivity of induced anti-M2e immunity was challenged for all 3 groups by intranasal infection with 10 LD50 of mouse adapted highly pathogenic Influenza A/Puerto Rico8/34 virus on day 60 after immunization with either of E277M2e-P2, CVJE, or Acam-FluA (
Thus, we demonstrate that viable chimeric flaviviruses can be engineered to display the M2e peptide on their E protein. The present data show that the same inserted peptide (e.g., M2e) may have different effects when inserted at different locations in the E protein. Furthermore, in order to characterize viruses into which epitopes have been inserted, propagation at 34° C. may be advantageous, enabling isolation of single viral clones which can be further characterized, for example by cDNA sequencing and immunization of animals and humans.
1All sequences in this table correspond to SEQ ID NO: 28, except otherwise indicated
2SEQ ID NO: 29
3SEQ ID NO: 30
4SEQ ID NO: 31
Campylobacter jejuni
Helicobacter pylori
Salmonella typhi
Vibrio cholerae
Clostridium difficile
Clostridium tetani
Streptococccus pyogenes
Bordetella pertussis
Neisseria meningitides
Neisseria gonorrhoea
Legionella neumophilus
Chlamydial spp.
Haemophilus spp.
Shigella spp.
Plasmodium spp.
Schistosoma spp.
Trypanosoma spp.
Toxoplasma spp.
Cryptosporidia spp.
Pneumocystis spp.
Leishmania spp.
The contents of all references cited above are incorporated herein by reference. Use of singular forms herein, such as “a” and “the,” does not exclude indication of the corresponding plural form, unless the context indicates to the contrary. Thus, for example, if a claim indicates the administration of “a” flavivirus, it can also be interpreted as covering administration of more than one flavivirus, unless otherwise indicated. Other embodiments are within the following claims.
This application claims priority under 35 U.S.C. §371 from international application PCT/US2008/001309, filed Jan. 31, 2008, which claims benefit of U.S. Provisional Patent Application No. 60/898,651, filed Jan. 31, 2007.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2008/001309 | 1/31/2008 | WO | 00 | 5/27/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/115314 | 9/25/2008 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5719051 | Mundt et al. | Feb 1998 | A |
| 6309650 | Kim et al. | Oct 2001 | B1 |
| 6497884 | Pletnev et al. | Dec 2002 | B1 |
| 6685948 | Zeng et al. | Feb 2004 | B1 |
| 6893866 | Westaway et al. | May 2005 | B1 |
| 8029802 | Guirakhoo et al. | Oct 2011 | B2 |
| 20030044773 | Kleanthous et al. | Mar 2003 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 02102828 | Dec 2002 | WO |
| WO 2006044857 | Apr 2006 | WO |
| WO 2006116182 | Nov 2006 | WO |
| Entry |
|---|
| Ebrahimi et al., Virus Genes, 2011, 42:1-9. |
| Kuno et al., In Vitro Cellular and Developmental Biology, Feb. 1989, 26(2):193-196. |
| Fiers et al., Virus Research, 2004, 103:173-176. |
| Wu et al., “Characterization of Immunity Induced by M2e of Influenza Virus,” Vaccine 25:8868-8873, 2007. |
| Bonaldo et al., “Expression of Foreign Protein Epitopes at the Surface of Recombinant Yellow Fever 17D Viruses Based on Three-Dimensional Modeling of its Envelope Protein,” Cell Biochem. Biophys. 44:313-324, 2006. |
| Jones et al., “Construction and Applications of Yellow Fever Virus Replicons,” Virology 331:247-259, 2005. |
| Kim et al., “Study on Persistent Infection of Japanese Encephalitis Virus Beijing-1 Strain in Serum-Free Sf9 Cell Cultures,” J. Microbiol. 42:25-31, 2004. |
| Wechuck et al., “Effect of Temperature, Medium Composition, and Cell Passage on Production of Herpes-Based Viral Vectors,” Biotechnology and Bioengineering 79:112-119, 2002. |
| Zou et al., “The Epitope Recognized by a Monoclonal Antibody in Influenza A Virus M2 Protein is Immunogenic and Confers Immune Protection,” Int. Immunopharmacol. 5:631-635, 2005. |
| XP-002582379, Database UniProtKB/TrEMBL, Q99DQ9 (online) “Polyprotein” Jun. 1, 2001. |
| Extended European Search Report from European Application No. EP 08799654, dated Jun. 9, 2010 (date of completion of search) and Jun. 17, 2010 (date of mailing of report). |
| International Preliminary Report on Patentability from International Application No. PCT/US2008/001309, dated Sep. 8, 2009. |
| Bonaldo et al., “Attenuation of Recombinant Yellow Fever 17D Viruses Expressing Foreign Protein Epitopes at the Surface,” J. Virol. 79:8602-8613, 2005. |
| Mandl et al., “Antigenic Structure of the Flavivirus Envelope Protein E at the Molecular Level, Using Tick-Borne Encephalitis Virus as a Model,” J. Virol. 63:564-571, 1989. |
| Wu et al., “Structural Basis of a Flavivirus Recognized by its Neutralizing Antibody,” J. Biol. Chem. 278:46007-46013, 2003. |
| International Search Report from International Application No. PCT/US2008/001309, dated May 28, 2009 (date of completion of search), and Jun. 10, 2009 (date of mailing of report). |
| Written Opinion from International Application No. PCT/US2008/001309 dated May 28, 2009 (date of completion of opinion), and Jun. 10, 2009 (date of mailing of opinion). |
| Number | Date | Country | |
|---|---|---|---|
| 20100255028 A1 | Oct 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60898651 | Jan 2007 | US |
