The invention provides attenuated Flavivirus vaccines, such as vaccines against Japanese encephalitis virus and West Nile virus, as well as methods of making and using these vaccines.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides recombinant Flaviviruses that include one or more membrane (M) protein mutations (e.g., substitutions, deletions, or insertions), such as mutations that attenuate the Flavivirus (e.g., mutations that decrease the viscerotropism/viremia of the Flavivirus), increase genetic stability of the Flavivirus during propagation in cell culture (e.g., manufacturing in serum free cultures), and/or increase vaccine virus yields. The Flaviviruses of the invention can be chimeric Flaviviruses, such as Flaviviruses that include capsid and non-structural proteins of a first Flavivirus (e.g., a yellow fever virus, such as YF 17D) and membrane and/or envelope proteins of a second Flavivirus (e.g., Japanese encephalitis virus, West Nile virus, a dengue virus (dengue-1, dengue-2, dengue-3, or dengue-4 virus), St. Louis encephalitis virus, Murray Valley encephalitis virus, tick-borne encephalitis virus, as well as any other Flavivirus that is a human/animal pathogen from the YF, JE, DEN, and TBE serocomplexes).

In the Flaviviruses of the invention, the mutation (e.g., substitution) can be in the transmembrane or ectodomain of membrane protein M. For example, the mutation can be in the region of amino acids 40-75 of the predicted membrane helix of the membrane protein M of the Flavivirus. As an example, the mutation can be a substitution of amino acid 60 of the membrane protein of a Flavivirus such as Japanese encephalitis virus (e.g., arginine to cysteine in the Japanese encephalitis virus M protein), or in a corresponding amino acid of another Flavivirus. Determination of which amino acid in a given Flavivirus "corresponds" to that of another Flavivirus can be carried out by standard amino acid sequence alignment, as is well known to those of skill in this art. As another example, the mutation can be a substitution of amino acid 66 of the membrane protein of a Flavivirus such as West Nile virus (e.g., a substitution of leucine with proline in the M protein of West Nile virus), or in a corresponding amino acid of another Flavivirus. In other examples, the mutation is at another membrane anchor amino acid, e.g., one or more amino acids selected from the group flanking the M66 residue, including positions 60, 61, 62, 63, 64, 65, and 66 of Japanese encephalitis virus or West Nile virus (or corresponding amino acids in other Flaviviruses) or other amino acid residues of the transmembrane domain.

We also provide for the first time evidence that the ectodomain of the M protein is of important functional significance, because a glutamine to proline change at the M5 residue increased the pH threshold of infection. Therefore, it can now be expected that Flavivirus attenuation can be achieved through amino acid changes or introduction of various deletions or insertions in the amino-terminal ectodomain, or surface part of the M protein, not only its C-terminal hydrophobic anchor. Thus, in other examples, the viruses of the invention include one or more mutations in the M protein ectodomain (residues 1-40) as described herein. This result is quite unexpected, given the lack of any known function of the mature M protein of Flaviviruses.

In addition to the membrane protein mutations noted above, in the case of chimeric Flaviviruses that include membrane and envelope proteins of a West Nile virus, the viruses of the invention can include one or more envelope protein mutations in amino acids selected from the group consisting of amino acids 107, 138, 176, 177, 224, 264, 280, 316, and 440. In other Flaviviruses, the mutations can be present in amino acids that correspond to these amino acids. As a specific example, the Flavivirus can include a mutation corresponding to mutation(s) in West Nile M protein amino acid 66 and E protein mutations at amino acids corresponding to West Nile virus amino acids 107, 316, and 440. In addition to the mutations described above, the Flaviviruses of the invention can also include one or more mutations in the hydrophobic pocket of the hinge region of the envelope protein, as described elsewhere herein. Further mutations that can be included in the viruses of the invention are mutations in the 3'UTR, the capsid protein, or other envelope protein regions, as described further below.

The invention also provides vaccine compositions that include the Flaviviruses described above and elsewhere herein and pharmaceutically acceptable carriers or diluents, as well as methods of inducing immune responses to Flaviviruses in patients by administration of such vaccine compositions. The patients treated according to such methods include those that do not have, but are at risk of developing, infection by the Flavivirus, as well as patients that are infected by the Flavivirus. Further, the invention includes the use of the Flaviviruses described herein in the prophylactic and therapeutic methods described herein, as well as in the manufacture of medicaments for these purposes.

The invention further provides methods of producing vaccines that include a Flavivirus as described herein, which involve introducing into the membrane protein of the Flavivirus a mutation that results in decreased viscerotropism/viremia, and/or increased genetic stability/yields. Further, the invention provides nucleic acid molecules (RNA or DNA) corresponding to the genomes of the Flaviviruses described herein (or the complements thereof), and methods of using such nucleic acid molecules to make the viruses of the invention.

The Flaviviruses of the invention are advantageous because, in having decreased virulence (shown, e.g., by decreased viscerotropism/viremia), they provide an additional level of safety, as compared to their non-mutated counterparts, when administered to patients. An additional advantage is that some mutations, such as the M-60 mutation in ChimeriVax.TM.-JE, preclude accumulation of undesirable mutations during vaccine manufacture that otherwise could compromise safety, and increase manufacturing yields. Additional advantages of these viruses are provided by the fact that they can include sequences of yellow fever virus strain YF17D (e.g., sequences encoding capsid and non-structural proteins), which (i) has had its safety established for >60 years, during which over 350 million doses have been administered to humans, (ii) induces a long duration of immunity after a single dose, and (iii) induces immunity rapidly, within a few days of inoculation. In addition, the vaccine viruses of the invention cause an active infection in the treated patients. As the cytokine milieu and innate immune response of immunized individuals are similar to those in natural infection, the antigenic mass expands in the host, properly folded conformational epitopes are processed efficiently, the adaptive immune response is robust, and memory is established.

The beneficial aspects of mutations in the M protein on vaccine safety and manufacture in cell culture are novel and unexpected, given the lack of any known function of the mature M protein of Flaviviruses.

DETAILED DESCRIPTION

The invention provides vaccines and methods for use in preventing and treating Flavivirus (e.g., Japanese encephalitis (JE) or West Nile (WN) virus) infection. The methods of the invention generally involve vaccination of subjects with a live, attenuated chimeric Flavivirus that consists of a first Flavivirus (e.g., yellow fever virus) in which one or more structural proteins (e.g., membrane and/or envelope proteins) have been replaced with those of a second Flavivirus (e.g., Japanese encephalitis (JE) and/or West Nile (WN) virus; also see below). The membrane proteins of the chimeras of the invention include one or more mutations, as is described further below. Also as is described below, structural proteins such as membrane and/or envelope proteins of other Flaviviruses can be used in place of those of Japanese encephalitis virus or West Nile virus in the chimeric viruses of the present invention. Further, the membrane protein mutations of the invention can also be used in intact, non-chimeric Flaviviruses (e.g., any of those listed herein), not including any replacements of structural proteins, and optionally with one or more additional mutations, such as those described herein.

A specific example of a chimeric virus that can be included in the vaccines of the invention is the human yellow fever vaccine strain, YF17D (e.g., YF17D-204 (YF-VAX.RTM., Sanofi-Pasteur, Swiftwater, Pa., USA; Stamaril.RTM., Sanofi-Pasteur, Marcy-L'Etoile, France; ARILVAX.TM., Chiron, Speke, Liverpool, UK; FLAVIMUN.RTM., Berna Biotech, Bern, Switzerland); YF17D-204 France (X15067, X15062); YF17D-204, 234 US (Rice et al., Science 229:726-733, 1985)), in which the membrane and envelope proteins have been replaced with the membrane and envelope proteins (including an M protein mutation, such as a substitution in M60, as described herein) of Japanese encephalitis virus. In another example, the YF 17D membrane and envelope proteins are replaced with those of a West Nile virus (including an M protein mutation, such as a substitution in M66, as described herein).

In other examples, another Flavivirus, such as a dengue virus (serotype 1, 2, 3, or 4), St. Louis encephalitis virus, Murray Valley encephalitis virus, yellow fever virus, including YF 17D strains, or any other Flavivirus, can provide the membrane and/or envelope proteins in such a chimeric virus. Additional Flaviviruses that can be attenuated according to the invention, whether as intact, non-chimeric viruses or as the source of membrane and/or envelope proteins in chimeras, include other mosquito-borne Flaviviruses, such as 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). Other yellow fever virus strains, e.g., YF17DD (GenBank Accession No. U 17066), YF17D-213 (GenBank Accession No. U17067; dos Santos et al., Virus Res. 35:35-41, 1995), and yellow fever virus 17DD strains described by Galler et al., Vaccines 16(9/10):1024-1028, 1998, can also be used as the backbone viruses into which heterologous structural proteins can be inserted according to the invention.

The viruses listed above each have some propensity to infect visceral organs. The viscerotropism of these viruses may cause dysfunction of vital visceral organs, such as observed in YF vaccine-associated adverse disease events, albeit very infrequently. The replication of virus in these organs can also cause viremia and thus contribute to invasion of the central nervous system. Decreasing the viscerotropism of these viruses by mutagenesis according to the present invention can thus reduce the abilities of the viruses to cause adverse viscerotropic disease and/or to invade the brain and cause encephalitis.

The mutations of the invention result in beneficial effects to the viruses, which can include, for example, increased attenuation, stability, and/or replication. The mutations are present in the membrane protein, e.g., in the transmembrane region or in the ectodomain of the membrane protein. For example, the mutations can be in amino acid 60 or 66 of the membrane protein and/or in other amino acids within the predicted transmembrane domain (e.g., in any one or more of amino acids 40-75), or in the N-terminal ectodomain of the M protein (e.g., M-5). As a specific example, membrane protein amino acid 60 (arginine in wild type Japanese Encephalitis virus) can be replaced with another amino acid, such as cysteine. A substitution from arginine to cysteine at position M-60 in the ChimeriVax.TM.-JE virus significantly reduced the viremia (viscerotropism) of the virus for humans in clinical trials in which variants of the vaccine with and without the M-60 mutation were tested (Tables 11A and 11B (see Original Patent)). In addition to cysteine, other amino acids, such as serine, threonine, glycine, methionine, etc., can substitute the wild type amino acid at position 60 of the membrane protein. In another example, membrane protein amino acid 66 (leucine in wild type West Nile virus) can be replaced with another amino acid, such as proline. In addition to proline, other hydrophobic amino acids, such as isoleucine, methionine, or valine, or small amino acids, such as alanine or glycine, can substitute the wild type amino acid at position 66 of the membrane protein. These mutations can also be present in corresponding amino acids of other Flaviviruses, as described herein.

As other examples of substitutions that can be made in membrane protein sequences, amino acids at positions 61, 62, 63, and/or 64 can be substituted, alone or in combination with each other, a mutation at position 60, a mutation at position 66, and/or another mutation(s). Examples of substitutions at these positions in the West Nile virus membrane protein sequence include: valine to alanine at position 61, valine to glutamic acid or methionine at position 62, phenylalanine to serine at position 63, and valine to isoleucine at position 64. These mutations can also be present in corresponding amino acids of other Flaviviruses, as described herein.

Examples of substitutions at these or surrounding positions in the JE virus membrane protein sequence include any of the remaining 20 amino acids with the expectation that a desired effect on viscerotropism and/or vaccine virus replication/stability in cell culture during manufacturing will be achieved. Other examples in chimeric or non-chimeric Flaviviruses include any amino acid substitutions, alone or in combinations, in the N-terminal ectodomain of the M protein composed of residues 1-.about.40 of the protein, as well as deletion(s) of various sizes (e.g., 1, 2, 3, 4, 5, etc., amino acids long) introduced into the ectodomain and/or the transmembrane domain of the M protein.

In addition to one or more of the membrane protein mutations noted above, the viruses of the invention can also include one or more additional mutations. For example, in the case of West Nile virus, such an additional mutation(s) can be in the region of position 107 (e.g., L to F), 316 (e.g., A to V), or 440 (e.g., K to R) (or a combination thereof) of the West Nile virus envelope protein. The mutations can thus be, for example, in one or more of amino acids 102-112, 138 (e.g., E to K), 176 (e.g., Y to V), 177 (e.g., T to A), 244 (e.g., E to G), 264 (e.g., Q to H), 280 (e.g., K to M), 311-321, and/or 435-445 of the West Nile envelope protein. As a specific example, using the sequence of West Nile virus strain NY99-flamingo 382-99 (GenBank Accession Number AF196835) as a reference, the lysine at position 107 can be replaced with phenylalanine, the alanine at position 316 can be replaced with valine, and/or the lysine at position 440 can be replaced with arginine. Examples of additional combinations of amino acids that can be mutated include are as follows: 176, 177, and 280; 176, 177, 244, 264, and 280; and 138, 176, 177, and 280. Further, these mutations can also be present in corresponding amino acids of other Flaviviruses, as described herein.

The ChimeriVax.TM.-JE vaccine already includes all of the above-noted SA14-14-2 specific mutations as it contains the SA14-14-2-specific JE envelope. Additional amino acid changes in the E protein can also be selected and introduced based on the knowledge of the structure/function of the E protein for additional attenuation (e.g., as described below). These mutations can also be present in corresponding amino acids of other Flaviviruses, as described herein.

In addition to the amino acids noted above, the substitutions can be made with other amino acids, such as amino acids that would result in conservative changes from those noted above. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, and leucine; aspartic acid, glutamic acid, asparagine, and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

The viruses of the invention (e.g., Japanese encephalitis and West Nile viruses, and chimeric Flaviviruses including membrane and envelope proteins from these or other flaviviruses) can also include in addition to the mutation(s) (e.g., membrane protein mutations) discussed above, one or more mutations in the hinge region or the hydrophobic pocket of the envelope protein, as such mutations have been shown to result in decreased viscerotropism (Monath et al., J. Virol. 76:1932-1943, 2002; WO 03/103571 A2; WO 05/082020; Guirakhoo et al., J. Virol. 78(18):9998-10008, 2004). The polypeptide chain of the envelope protein folds into three distinct domains: a central domain (domain I), a dimerization domain (domain II), and an immunoglobulin-like module domain (domain III). The hinge region is present between domains I and II and, upon exposure to acidic pH, undergoes a conformational change (hence the designation "hinge") that results in the formation of envelope protein trimers that are involved in the fusion of viral and endosomal membranes, after virus uptake by receptor-mediated endocytosis. Prior to the conformational change, the proteins are present in the form of dimers.

Numerous envelope amino acids are present in the hinge region including, for example, amino acids 48-61, 127-131, and 196-283 of yellow fever virus (Rey et al., Nature 375:291-298, 1995). Any of these amino acids, or closely surrounding amino acids (and corresponding amino acids in other Flavivirus envelope proteins), can be mutated according to the invention, and tested for attenuation. Of particular interest are amino acids within the hydrophobic pocket of the hinge region. As a specific example, it has been shown that substituting envelope protein amino acid 204 (K to R), which is in the hydrophobic pocket of the hinge region, in a chimeric Flavivirus including dengue 1 envelope protein sequences inserted into a yellow fever virus vector results in attenuation (Guirakhoo et al., J. Virol. 78:9998-10008, 2004). This substitution leads to an alteration in the structure of the envelope protein, such that intermolecular hydrogen bonding between one envelope monomer and another in the wild type protein is disrupted and replaced with new intramolecular interactions within monomers. This observation led to a proposal that the attenuation resulting from this substitution is due to these new interactions, which change the structure of the protein in the pre-fusion conformation, most likely by altering the pH threshold that is required for fusion of viral membrane with the host cell, and provides a basis for the design of further attenuated mutants in which additional substitutions are used to increase intramolecular interactions in the hydrophobic pocket, leading to attenuation. Examples of such mutations/substitutions that can be made in the hydrophobic pocket, and included in the viruses of the invention, include substitutions in E202K, E204K, E252V, E253L, E257E, E258G, and E261H (and corresponding substitutions in other Flaviviruses). Any amino acid changes in the corresponding region of the E protein of JE and WN viruses can be designed and incorporated based on the knowledge of homologous protein structure.

The E gene contains functional domains within which amino acid changes may affect function and thereby reduce virulence, as described by Hurrelbrink and McMinn (Adv. Virus Dis. 60:1-42, 2003). The functional regions of the E protein in which mutations may be inserted that, together with the membrane deletions/mutations described in the present application, may result in an appropriately attenuated vaccine include: a) the putative receptor binding region on the external surface of domain III, b) the molecular hinge region between domains I and II, which determines the acid-dependent conformational changes of the E protein in the endosome and reduce the efficiency of virus internalization; c) the interface ofprM and E proteins, a region of the E protein that interfaces with prM following the rearrangement from dimer to trimer after exposure to low pH in the endosome; d) the tip of the fusion domain of domain II, which is involved in fusion to the membrane of the endosome during internalization events; and e) the stem-anchor region, which is also functionally is involved in conformational changes of the E protein during acid-induced fusion events.

Additional attenuating mutations that can be included with one or more membrane protein mutations in the viruses of the invention include mutations in the 3'untranslated region of the yellow fever virus backbone. The organization of the 3'UTR of a yellow fever virus vaccine strain, YF 17D, which is shared by all ChimeriVax.TM. viruses, is shown in FIG. 1A (see Original Patent). It includes in order from the 3' end (i) a 3'-extreme stem-and-loop structure that has been hypothesized to function as a promoter for minus-strand RNA synthesis and is conserved for all Flaviviruses, (ii) two conserved sequence elements, CS1 and CS2, which share a high degree of nucleotide sequence homology with all mosquito-borne Flaviviruses, and (iii) unique for West African yellow fever virus strains, including the YF 1 7D vaccine virus, three copies of a repeat sequence element (RS) located in the upstream portion of the 3'UTR (Chambers et al., Annu. Rev. Microbiol. 44:649-688, 1990). The 3'UTR also includes numerous stem-loop structures, such as those in the non-conserved region downstream from the RS elements, as depicted in FIG. 1B (see Original Patent). 3'UTR mutations that can be included in the viruses of the invention generally are short, attenuating deletions of, for example, less than 30 nucleotides (e.g., 1, 2, 3, etc., and up to 29 (e.g., 2-25, 3-20, 4-15, 5-10, or 6-8 nucleotides in length); U.S. Patent Application Nos. 60/674,546 and 60/674,415). In some examples, the short 3'UTR deletions are designed to destabilize the secondary structure of one or more of the stem structures in the 3'UTR. In addition to deletions, mutations in such structures can also include substitutions that similarly result in stem structure destabilization. In certain examples, the stem-loop structures that are subject to the mutations are present in non-conserved regions of the 3'UTR or in conserved regions that can tolerate such mutations (e.g., in CS2). For example, the stem destabilizing mutations can be present in any one or more of the predicted stem structures shown in FIG. 1B, which shows four examples of such deletions (dA, dB, dC, and dD). Thus, in addition to these specific examples, other examples of 3'UTR mutations in yellow fever virus include mutations that comprise, e.g., 1-2, 3-8, 4-7, or 5-6 nucleotides of the following stem sequences, which are shown in FIG. 1B as read from 5' to 3': TGGAG, CTCCA, GACAG, TTGTC, AGTTT, GGCTG, CAGCC, AACCTGG, TTCTGGG, CTACCACC, GGTGGTAG, GGGGTCT, AGACCCT, AGTGG, and TTGACG. These mutations can also be present in corresponding amino acids of other Flaviviruses, as described herein.

In addition to stem destabilizing mutations, other short deletions in the 3'UTR can also be included with one or more membrane (and possibly other) mutations in the viruses of the invention. For example, the previously described .DELTA.30 mutation (Men et al., J. Virol. 70:3930-3937, 1996; U.S. Pat. No. 6,184,024 B1) or mutations that fall within this sequence can be used. Thus, for example, the invention includes any viable deletions that are 1, 2, 3, etc., and up to 29 (e.g., 1-25, 2-20, 3-15, 4-14, 5-13, 6-12, 7-11, 8-10, or 9) nucleotides in length within this region. As a specific example, viruses of the invention can include deletion d7, in which the following nucleotides from this region in YF17D are deleted: nucleotides 345-351 (AAGACGG; numbering from the 1.sup.st nucleotide of the 3'UTR, after the UGA termination codon of the viral ORF; FIG. 1A). Mutations that include deletion of, for example, 1, 2, 3, 4, or 5 additional nucleotides from the 3' or 5' end of this sequence are also included in the invention. In other examples, short deletions in conserved sequences CS1 and CS2 are included in the invention. These mutations can include deletion of, e.g., 1-29, 2-25, 3-20, 4-15, 5-10, or 6-8 nucleotides of these sequences. As two specific examples, nucleotides 360-364 (GGTTA; CS2d5; FIG. 1A) and/or nucleotides 360-375 (GGTTAGAGGAGACCCT (SEQ ID NO:17); CS2d16; FIG. 1A) are deleted from CS2 of the YF17D-specific 3'UTR. Mutations that include deletion of, for example, 1, 2, 3, 4, or 5 additional nucleotides from the 3' or 5' end of this sequence can also be used. For other flavivirus 3'UTRs, similar mutations can be made, based on the secondary structures of the 3 'UTR's. Predictions of secondary structures of 3'UTR of other flaviviruses have been published, e.g., for dengue, Kunjin, and TBE (see, e.g., Proutski et al., Virus Res. 64:107-123, 1999) and HCV (see, e.g., Kolykhalov et al., J. Virol. 70:3363-3371, 1996). Further, numerous 3'UTR nucleotide sequences for many strains of flaviviruses representing all four major serocomplexes (YF, JE, dengue, and TBE) are available from GenBank. Sequences of additional strains can be determined by virus sequencing. The secondary structures of these sequences can be easily predicted using standard software (e.g., mfold or RNAfold programs) to reveal potential stem-loop structures that can be subject to mutagenesis.

It should be noted that the true secondary structures of the 3'UTRs of Flaviviruses, including YF 17D virus, are unknown because there are no available methods to experimentally prove their existence in the context of whole viruses, and therefore published predictions, e.g., the one predicted for YF 17D by Proutski and co-workers (FIG. 1B), may be incorrect. Many alternative structures can be predicted to form in a relatively long RNA molecule (Zuker et al., N. A. R. 19:2707-2714, 2001), and it is possible that different structures (in plus or minus strands) form and function at different steps of the viral life cycle. True structures can be influenced by the formation of various pseudoknots (Olsthoorn et al., RNA 7:1370-1377, 2001) and long range RNA interactions (e.g., RNA cyclization and other interactions (Alvarez et al., J. Virol. 79:6631-6643, 2005)), as well as possible RNA interactions with host and viral proteins. To further complicate interpretation of published results of theoretical computer predictions, manual operations are often used, such as initial folding of partial sequences with subsequent forcing of initially predicted structures into structures of longer RNA sequences, the artificial use of N's during initial folding steps, and subjective selection of preferred structure elements (e.g., Mutebi et al., J. Virol. 78:9652-9665, 2004). To this end, we folded the 3'UTR RNA sequence of YF 17D using the commonly used Zuker's prediction algorithm. The predicted optimal structure is shown in FIG. 1C (see Original Patent), which differs from the Proutsky prediction shown in FIG. 1B. It is important that the small deletions dA, dB, dC, dD, d7, and d14 in FIGS. 1A and 1B generally destabilized the predicted native YF 17D optimal (FIG. 1C) and suboptimal structures. An example of one such altered optimal structure (for the dC mutant) is shown in FIG. 1D (see Original Patent). In contrast, the CS2d5 and CS2d16 deletions (FIGS. 1A and 1B) did not noticeably change the optimal native structure, indicating that these deletions may attenuate the virus (attenuation was demonstrated in the hamster model for ChimeriVax.TM.-WN) by virtue of altering the sequence of CS2 per se rather than the 3 'UTR structure, or alternatively by altering some suboptimal structures. Thus, even though some of the deletions were designed based on the Proutski structure prediction (FIG. 1B), their true effect may be due to destabilizing different structure elements than the predicted stem-loops in FIG. 1B.

Additional mutations that can be included with membrane protein (and possibly other) mutations in the viruses of the invention are short deletion (e.g., deletions of 1, 2, 3, or 4 amino acids) mutations within the capsid protein. Examples of such mutations, provided in reference to the YF 17D virus capsid protein, include viable deletions affecting Helix I of the protein (see FIG. 2A). A specific example of such a mutation is mutation C2, which includes a deletion of amino acids PSR from Helix I (FIG. 2A). Other short mutations in this region (as well as corresponding mutations in other Flavivirus sequences) can be tested for viability and attenuation, and can also be used in the invention. Capsid protein sequences of other flaviviruses have been published, e.g., for TBE, W N, Kunjin, J E, and dengue viruses (e.g., Pletnev et al., Virology 174:250-263, 1990).

The following are specific examples of 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, that can be used to make viruses of the invention: Chimeric Yellow Fever 17D/Dengue Type 2 Virus (YF/DEN-2; ATCC accession number ATCC VR-2593) and Chimeric Yellow Fever 17D/Japanese Encephalitis SA14-14-2 Virus (YF/JE A1.3; ATCC accession number ATCC VR-2594). Details of making chimeric viruses that can be used in the invention are provided, for example, in U.S. Pat. No. 6,696,281 B1; international applications PCT/US98/03894 (WO 98/37911) and PCT/US00/32821 (WO 01/39802); and Chambers et al., J. Virol. 73:3095-3101, 1999, and are also provided below. These methods can be modified for use in the present invention by including a step of introducing one or more mutations as described herein into inserted sequences (e.g., Japanese encephalitis virus or West Nile virus membrane protein or other sequences). Methods that can be used for producing viruses in the invention are also described in PCT/US03/01319 (WO 03/060088 A2; also see below).

Mutations can be made in the viruses of the invention using standard methods, such as site-directed mutagenesis. One example of the type of mutation present in the viruses of the invention is substitutions, but other types of mutations, such as deletions and insertions, can be used as well. In addition, as is noted above, the mutations can be present singly or in the context of one or more additional mutations, whether within the membrane protein itself or in any combination of, e.g., 3 'UTR, capsid, or envelope sequences.

The viruses (including chimeras) of the present invention 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, chick embryos, or diploid cell lines, from which (or the supernatants of which) progeny virus can then be purified. Another method that can be used to produce the viruses employs heteroploid cells, such as Vero cells (Yasumura et al., Nihon Rinsho 21:1201-1215, 1963). In this method, 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, and harvested virus is treated with a nuclease (e.g., an endonuclease that degrades both DNA and RNA, such as Benzonase.TM.; U.S. Pat. No. 5,173,418). In the case of Benzonase.TM., 15 units/mL can be used, and the conditioned medium refrigerated at 2-8.degree. C. for about 16 or more hours to allow for digestion of nucleic acids. The nuclease-treated virus is then concentrated (e.g., by use of ultrafiltration using a filter having a molecular weight cut-off of, e.g., 500 kDa (e.g., a Pellicon-2 Mini unltrafilter cassette)), diafiltered against MEME without phenol red or FBS, formulated by the addition of lactose, and filtered into a sterile container. Details of this method are provided in WO 03/060088 A2. Further, cells used for propagation of viruses of the invention can be grown in serum free medium, as described below.

The viruses of the invention can be administered as primary prophylactic agents in those at risk of infection, or can be used as secondary agents for treating infected patients. Because the viruses are attenuated, they are particularly well-suited for administration to "at risk individuals" such as the elderly, children, or HIV infected persons. The vaccines can also be used in veterinary contexts, e.g., in the vaccination of horses against West Nile virus infection, or in the vaccination of domestic pets (e.g., cats, dogs, and birds), livestock (e.g., sheep, cattle, pigs, birds, and goats), and valuable animals such as rare birds. Further, the vaccines of the invention can include a virus, such as a chimeric virus, including a particular mutation (e.g., the M5, M60, and/or M66 mutation), in a mixture with viruses lacking such mutations.

Formulation of the viruses of the invention can be carried out using methods that are standard in the art. Numerous pharmaceutically acceptable solutions for use in vaccine preparation are well known and can readily be adapted for use in the present invention by those of skill in this art (see, e.g., Remington 's Pharmaceutical Sciences (18.sup.th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, Pa.). In two specific examples, the viruses are formulated in Minimum Essential Medium Earle's Salt (MEME) containing 7.5% lactose and 2.5% human serum albumin or MEME containing 10% sorbitol. However, the viruses can simply be diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline. In another example, the viruses 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 vaccines of the invention can be administered using methods that are well known in the art, and appropriate amounts of the vaccines to be administered can readily be determined by those of skill in the art. What is determined to be an appropriate amount of virus to administer can be determined by consideration of factors such as, e.g., the size and general health of the subject to whom the virus is to be administered. For example, the viruses of the invention can be formulated as sterile aqueous solutions containing between 10.sup.2 and 10.sup.8, e.g., 10.sup.3 to 10.sup.7 or 10.sup.4 to 10.sup.6, 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. In addition, because Flaviviruses may be capable of infecting the human host via 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 viruses can be administered by mucosal (e.g., oral) routes as well. Further, the vaccines of the invention can be administered in a single dose or, optionally, administration can involve the use of a priming dose followed by one or more booster doses that are administered, e.g., 2-6 months later, as determined to be appropriate by those of skill in the art.

Optionally, adjuvants that are known to those skilled in the art can be used in the administration of the viruses of the invention. Adjuvants that can be used to enhance the immunogenicity of the viruses 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 virus 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 viruses. Thus, genes encoding cytokines, such as GM-CSF, IL-2, IL-1 2, 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. Additional adjuvants that can optionally be used in the invention include toll-like receptor (TLR) modulators.

In the case of dengue viruses and/or chimeric Flaviviruses including membrane and envelope proteins of a dengue virus, against which optimal vaccination can involve the induction of immunity against all four of the dengue serotypes, the viruses of the invention can be used in the formulation of tetravalent vaccines. Any or all of the viruses used in such tetravalent formulations can include one or more mutations that decrease viscerotropism, as is described herein. The viruses can be mixed to form tetravalent preparations at any point during formulation, or can be administered in series. In the case of a tetravalent vaccine, equivalent amounts of each virus may be used. Alternatively, the amounts of each of the different viruses present in the administered vaccines can vary (WO 03/101397 A2).

The invention also includes nucleic acid molecules (e.g., RNA or DNA (e.g., cDNA) molecules) that correspond to the genomes of the viruses of the invention as described herein, or the complements thereof. These nucleic acid molecules can be used, for example, in methods of manufacturing the viruses of the invention. In such methods, a nucleic acid molecule corresponding to the genome of a virus is introduced into cells in which the virus can be produced and replicate (e.g., primary cells, chick embryos, diploid cell lines, or heteroploid cell lines (e.g., Vero cells)), and from which (or the supernatants of which) progeny virus can then be purified. These methods can further include virus purification steps, as is known in the art.

As is noted above, details of making chimeric viruses that can be used in the invention are provided, for example, in U.S. Pat. No. 6,696,281 B1; international applications PCT/US98/03894 (WO 98/37911) and PCT/US00/32821 (WO 01/39802); and Chambers et al., J. Virol. 73:3095-3101, 1999. Details of the construction of a chimeric Flavivirus including pre-membrane and envelope proteins of Japanese encephalitis virus (or West Nile virus), and capsid and non-structural proteins of yellow fever virus, are provided as follows. These methods can readily be adapted by those of skill in the art for use in constructing chimeras including the mutations described herein, as well as chimeras including other pre-membrane and envelope sequences.

Briefly, derivation of a YF/JE chimera can involve the following. YF genomic sequences are propagated in two plasmids (YF5'3'IV and YFM5.2), which encode the YF sequences from nucleotides 1-2,276 and 8,279-10,861 (YF5'3'IV) and from 1,373-8,704 (YFM5.2) (Rice et al., The New Biologist 1:285-296, 1989). Full-length cDNA templates are generated by ligation of appropriate restriction fragments derived from these plasmids. YF sequences within the YF5'3'IV and YFM5.2 plasmids are then replaced by the corresponding JE sequences from the start of the prM protein (nucleotide 478, amino acid 128) through the E/NS1 cleavage site (nucleotide 2,452, amino acid 817).

Clones of authentic JE structural protein genes were generated from the JE SA14-14-2 strain (JE live, attenuated vaccine strain; JE SA14-14-2 virus is available from the Centers for Disease Control, Fort Collins, Colo. and the Yale Arbovirus Research Unit, Yale University, New Haven, Conn., which are World Health Organization-designated Reference Centers for Arboviruses in the United States). JE SA14-14-2 virus at passage level PDK-5 was obtained and passaged in LLC-MK.sub.2 cells to obtain sufficient amounts of virus for cDNA cloning. The strategy used involved cloning the structural region in two pieces that overlap at an NheI site (JE nucleotide 1,125), which can then be used for in vitro ligation.

RNA was extracted from monolayers of infected LLC-MK.sub.2 cells and first strand synthesis of negative sense cDNA was carried out using reverse transcriptase with a negative sense primer (JE nucleotide sequence 2,456-71) containing nested XbaI and NarI restriction sites for cloning initially into pBluescript II KS(+), and subsequently into YFM5.2(NarI), respectively. First strand cDNA synthesis was followed by PCR amplification of the JE sequence from nucleotides 1,108-2,471 using the same negative sense primer and a positive sense primer (JE nucleotides sequence 1,108-1,130) containing nested XbaI and NsiI restriction sites for cloning into pBluescript and YFM5.2(NarI), respectively. JE sequences were verified by restriction enzyme digestion and nucleotide sequencing. The JE nucleotide sequence from nucleotides 1 to 1,130 was derived by PCR amplification of negative strand JE cDNA using a negative sense primer corresponding to JE nucleotides 1,116 to 1,130 and a positive sense primer corresponding to JE nucleotides 1 to 18, both containing an EcoRI restriction site. PCR fragments were cloned into pBluescript and JE sequences were verified by nucleotide sequencing. Together, this represents cloning of the JE sequence from nucleotides 1-2,471 (amino acids 1-792).

To insert the C terminus of the JE envelope protein at the YF E/NS1 cleavage site, a unique NarI restriction site was introduced into the YFM5.2 plasmid by oligonucleotide-directed mutagenesis of the signalase sequence at the E/NS1 cleavage site (YF nucleotides 2,447-2,452, amino acids 816-817) to create YFM5.2(NarI). Transcripts derived from templates incorporating this change were checked for infectivity and yielded a specific infectivity similar to the parental templates (approximately 100 plaque-forming units/250 nanograms of transcript). The JE sequence from nucleotides 1,108 to 2,471 was subcloned from several independent PCR-derived clones of pBluescript/JE into YFM5.2(Narl) using the unique NsiI and NarI restriction sites. YF5'3'IV/JE clones containing the YF 5' untranslated region (nucleotides 1-118) adjacent to the JE prM-E region were derived by PCR amplification.

To derive sequences containing the junction of the YF capsid and JE prM, a negative sense chimeric primer spanning this region was used with a positive sense primer corresponding to YF5'3'IV nucleotides 6,625-6,639 to generate PCR fragments that were then used as negative sense PCR primers in conjunction with a positive sense primer complementary to the pBluescript vector sequence upstream of the EcoRI site, to amplify the JE sequence (encoded in reverse orientation in the pBluescript vector) from nucleotide 477 (N-terminus of the prM protein) through the NheI site at nucleotide 1,125. The resulting PCR fragments were inserted into the YF5'3'IV plasmid using the NotI and EcoRI restriction sites. This construct contains the SP6 promoter preceding the YF 5'-untranslated region, followed by the sequence: YF (C) JE (prM-E), and contains the NheI site (JE nucleotide 1,125) required for in vitro ligation.

To use the NheI site within the JE envelope sequence as a 5' in vitro ligation site, a redundant NheI site in the YFM5.2 plasmid (nucleotide 5,459) was eliminated. This was accomplished by silent mutation of the YF sequence at nucleotide 5,461 (T C; alanine, amino acid 1820). This site was incorporated into YFM5.2 by ligation of appropriate restriction fragments and introduced into YFM5.2(NarI)/JE by exchange of an NsiI/NarI fragment encoding the chimeric YF/JE sequence.

To create a unique 3' restriction site for in vitro ligation, a BspEI site was engineered downstream of the AatII site normally used to generate full-length templates from YF5'3'IV and YFM5.2. (Multiple AatII sites are present in the JE structural sequence, precluding use of this site for in vitro ligation.) The BspEI site was created by silent mutation of YF nucleotide 8,581 (A C; serine, amino acid 2,860), and was introduced into YFM5.2 by exchange of appropriate restriction fragments. The unique site was incorporated into YFM5.2/JE by exchange of the XbaI/SphI fragment, and into the YF5'3'IV/JE(prM-E) plasmids by three-piece ligation of appropriate restriction fragments from these parent plasmids and from a derivative of YFM5.2 (BspEI) deleting the YF sequence between the EcoRI sites at nucleotides 1 and 6,912.

cDNA from a clone of the JE Nakayama strain, which has been extensively characterized in expression experiments and for its capacity to induce protective immunity (see, e.g., Mclda et al., Virology 158:348-360, 1987; the J E Nakayama strain is available from the Centers for Disease Control, Fort Collins, Colo., and the Yale Arbovirus Research Unit, Yale University, New Haven, Conn.), was also used in the construction of chimeric flaviviruses. The Nakayama cDNA was inserted into the YF/JE chimeric plasmids using available restriction sites (HindIII to PvuII and BpmI to MunI) to replace the entire prM-E region in the two plasmid system except for a single amino acid, serine, at position 49, which was left intact in order to utilize the NheI site for in vitro ligation.

Procedures for generating full-length cDNA templates are essentially as described in Rice et al. (The New Biologist 1:285-96, 1989). In the case of chimeric templates, the plasmids YF5'3'IV/JE (prM-E) and YFM5.2/JE are digested with NheI/BspEI and in vitro ligation is performed using 300 nanograms of purified fragments in the presence of T4 DNA ligase. The ligation products are linearized with XhoI to allow run-off transcription. SP6 transcripts are synthesized using 50 nanograms of purified template, quantitated by incorporation of .sup.3H-UTP, and integrity of the RNA is verified by non-denaturing agarose gel electrophoresis. Yields range from 5 to 10 micrograms of RNA per reaction using this procedure, most of which is present as full-length transcripts. Transfection of RNA transcripts in the presence of cationic liposomes is carried out as described by Rice et al. (supra) for YF 17D, to generate the chimeric viruses.

In the case of chimeric flaviviruses including West Nile virus and yellow fever virus sequences, the two-plasmid system described above can also be used. In one example, the West Nile (WN) virus prM and E genes used were cloned from WNV flamingo isolate 383-99, sequence GenBank accession number AF196835. Virus prME cDNA was obtained by RT-PCR (XL-PCR Kit, Perkin Elmer). The 5' end of WN prM gene was cloned precisely at the 3 'end of the YF 17D capsid gene by overlap-extension PCR using Pwo polymerase (Roche). The 3' end of the E gene was also cloned precisely at the 5'end of the YF NS1 coding sequence by overlap-extension PCR. Silent mutations were introduced into the sequence of prM and E to create unique restriction sites Bsp EI and Eag I. Digestion of the two plasmids with these enzymes generated DNA fragments that were gel purified and ligated in vitro to produce a full-length chimeric cDNA. The cDNA was linearized with Xho I to facilitate in vitro transcription by SP6 polymerase (Epicentre). The RNA product was introduced into eukaryotic cell lines permissive for viral RNA translation and replication of the virus. As with the YF/JE chimera, described above, mutations of the invention can be introduced into YF/WN chimeras as described herein, using standard methods.
 

Claim 1 of 39 Claims

1. An attenuated chimeric flavivirus comprising a yellow fever virus wherein the membrane and envelope proteins of the yellow fever virus have been replaced with the membrane and envelope proteins of a Japanese encephalitis virus, wherein the transmembrane domain of the membrane protein of the chimeric flavivirus comprises a mutation, wherein the mutation is a substitution of amino acid 60 of the membrane protein of a Japanese encephalitis virus.

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