High yield yellow fever virus strain with increased propagation in cells

Abstract

The invention provides a an inactive, non-replicating vaccine comprising whole virion, chemically inactivated Yellow Fever virus which is inactivated using a method that ensures preservation of critical, neutralizing epitopes. The Yellow Fever virus has been adapted to propagate in cells to higher yields than the unadapted virus. The invention also provides methods for preventing Yellow Fever viral infection.

Claims

1. A modified Yellow Fever virus strain comprising a nucleic acid sequence having mutations relative to the nucleic acid sequence of unmodified Yellow Fever virus, wherein said mutations comprise: a mutation in the nucleic acid sequence encoding the NS1 protein of the virus in the codon for the amino acid at position 317 wherein the mutation results in a codon change from threonine to isoleucine; a mutation in the nucleic acid sequence encoding the NS2A protein of the virus in the codon for the amino acid position 170 wherein the mutation results in a codon change from phenylalanine to leucine, and optionally a mutation in the nucleic acid sequence encoding the NS4B protein of the virus in the codon for the amino acid at position 113 wherein the mutation results in a codon change from isoleucine to methionine, and said mutations are in further combination with a mutation of the nucleic acid sequence encoding the envelope protein of the virus in the codon for the amino acid at position 160 wherein the mutation results in a codon change from lysine to arginine, wherein said modified Yellow Fever virus strain has increased propagation in Vero cells and a higher yield in the conditioned medium of a Vero cell culture relative to unmodified Yellow Fever virus.

2. An inactivated Yellow Fever virus comprising the modified Yellow Fever virus of claim 1.

3. A vaccine comprising the inactivated virus of claim 2.

4. A method for inducing an immune response to Yellow Fever virus in a subject, the method comprising administering the vaccine of claim 3 to the subject.

5. The method according to claim 4, wherein the subject is at risk of developing, but does not have, Yellow Fever virus infection.

6. A method for making a vaccine comprising culturing cells infected with a modified Yellow Fever virus strain of claim 1, purifying the virus to generate a live virus bulk, and inactivating said virus live bulk.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic representation of the passage history of Vero cells during the manufacture of the disclosed yellow fever vaccine.

(2) FIG. 2 is a schematic representation of the preparation of the virus seeds.

(3) FIG. 3A is a schematic of the process used for 10 serial passages (P1 through P10) to modify the nucleotide sequence of the viral genome virus to develop a seed virus with enhanced growth in Vero cells for preparation of an inactivated Yellow Fever virus candidate.

(4) FIG. 3B is a graphical representation of the virus replication for passage one (P1) and passage 11 (P11) of the initial experiment, in which P11 virus differs from P1 by a single mutation at E160 (lys.fwdarw.arg)

(5) FIG. 3C is a graphical representation of a repeat passaging study of passage one (Bp1, Cp1) and passage 11 (B-p11, C-p11) virus performed in a series of experiments: Series B and C.

(6) FIG. 4A-L depicts the consensus alignment of the P1 and P11 nucleic acid sequences. The starting nucleic acid sequence, P1, is identified herein as SEQ ID NO: 1. A comparison of the P1 passage and the P11 passage revealed a genetic mutation at nucleotide residue #211 of SEQ ID NO: 1, and a second mutation at nucleotide residue #1452 of SEQ ID NO: 1. Thus, P1 consensus corresponds to SEQ ID NO.1; P11 consensus corresponds to SEQ ID NO. 2 having the codon mutation at envelope protein amino acid position 160.

(7) FIG. 5A-J depicts the amino acid sequence of P1 and P11, with the Series B-P1 and Series B3-P11 amino acid sequences from the repeat passaging study. The amino acid sequence for P1 is identified herein as SEQ ID NO: 3. A comparison of the amino acid sequence for P1 and that of P11 (SEQ ID NO. 4) revealed a mutation at amino acid residue 160 of the envelope protein (E160) (amino acid 445 of the P1 amino acid sequence in FIG. 5B). Series B-P1 and Series B3-P11 present partial amino acid sequences from the repeat passaging study. The amino acid sequence for B-P1 is identified herein as SEQ ID NO: 5. A comparison of the amino acid sequence for B-P1 and that of B3-P11 (SEQ ID NO. 6) revealed a mutation at amino acid residue 160 of the envelope protein (E160) in B3-P11 (amino acid 445 of the P1 amino acid sequence).

(8) FIG. 6 depicts the comparative 50% plaque reduction neutralization test (PRNT50) titers between treatment groups of BALB/c and CD-1 strain mice in a preliminary mouse study (M-9003-002) of the efficacy of inactivated Yellow Fever vaccine.

(9) FIG. 7 is a graphical representation of PRNT50 antibody titers for the preliminary mouse study (M-9003-002).

(10) FIG. 8A-EE depicts the consensus alignment of the P1, B-P1, C-P1, B3-P11 and C1-P11 nucleic acid sequences. The nucleic acid sequence, P1, is identified herein as SEQ ID NO: 15. The nucleic acid sequence, B-P1, is identified herein as SEQ ID NO: 9. The nucleic acid sequence, B3-P11, is identified herein as SEQ ID NO: 11. The nucleic acid sequence, C-P1, is identified herein as SEQ ID NO: 10. The nucleic acid sequence, C1-P11, is identified herein as SEQ ID NO: 12. A comparison of the B-P1 passage and the B3-P11 passage revealed a genetic mutation in B3-P11 at nucleotide residue #1452 of SEQ ID NO: 15, a second mutation in B3-P11 at nucleotide residue #3402 of SEQ ID NO: 15, and a third mutation in B3-P11 at nucleotide residue #4016 of SEQ ID NO: 15. A comparison of the C-P1 passage and the C1-P11 passage revealed a genetic mutation in C1-P11 at nucleotide residue #7225 of SEQ ID NO: 15. SEQ ID NO: 11 corresponds to B3-P11, and has the codon mutations at envelope protein amino acid position 160, non-structural protein 1 amino acid position 317, and non-structural protein 2A amino acid position 170. SEQ ID NO.12 corresponds to C1-P11, and has the codon mutation at non-structural protein 4B amino acid position 113.

(11) FIG. 9A-F depicts the amino acid sequence of B-P1 and B3-P11 from the repeat passaging study. The amino acid sequence for B-P1 is identified herein as SEQ ID NO: 13. A comparison of the amino acid sequence for B-P1 and that of B3-P11 (SEQ ID NO: 7) revealed a mutation at amino acid residue 160 of the envelope protein (E160) (amino acid 445 in FIG. 9A), a mutation at amino acid residue 317 of the non-structural protein 1 (NS1-317) (amino acid 1095 in FIG. 9B), and a mutation at amino acid residue 170 of the non-structural protein 2A (NS2A-170) (amino acid 1300 in FIG. 9C). Series B-P1 and Series B3-P11 present complete amino acid sequences from the repeat passaging study.

(12) FIG. 10A-F depicts the amino acid sequence of C-P1 and C1-P11 from the repeat passaging study. The amino acid sequence for C-P1 is identified herein as SEQ ID NO: 14. A comparison of the amino acid sequence for C-P1 and that of C1-P11 (SEQ ID NO: 8) revealed a mutation at amino acid residue 113 of the non-structural protein 4B (NS4B-113) (amino acid 2369 in FIG. 10E). Series C-P1 and Series C1-P11 present complete amino acid sequences from the repeat passaging study.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(13) A description of preferred embodiments of the invention follows. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. At the outset, the invention is described in its broadest overall aspects, with a more detailed description following. The features and other details of the compositions and methods of the invention will be further pointed out in the claims.

(14) Overview of Approach and Benefits

(15) The invention relates to compositions and methods for use in preventing Yellow Fever virus infection. Disclosed herein is a method of producing an inactivated Yellow Fever virus candidate, the method comprising the serial passage of the YF 17D virus (i.e., an unadapted virus) in certified African green monkey kidney cells (VERO) to increase the titer to yield a sufficient antigenic mass to induce a protective immune response and/or modify the nucleotide sequence of the viral genome. This method has been repeated and shown to be reproducible.

(16) One embodiment of the invention is a modified Yellow Fever (YF) virus that will grow to high titers in Vero cells. Another embodiment of the invention is a vaccine comprising a whole virion, chemically inactivated Yellow Fever (YF) virus prepared from serum-free conditioned medium from Vero cells infected with 17D virus. In one embodiment of the invention, the virus has been purified from host cell DNA and proteins by depth filtration, ultrafiltration, diafiltration, and chromatographic separation. The method is described in U.S. Application Ser. No. 61/228,026 filed on Jul. 23, 2009, and its corresponding International Application No. PCT/US2010/043013 filed on Jul. 23, 2010, which are each incorporated herein by reference. The purified virus may be inactivated by using a method that ensures preservation of critical, neutralizing epitopes. For example, the virus can be inactivated using formalin, heat, UV, gamma irradiation or beta-propiolactone. A purified, inactivated virus may be formulated with an adjuvant, such as adsorbed to aluminum hydroxide adjuvant, and stored as a liquid at temperatures of from about 2 degrees Celsius (2 C.) to about 8 degrees Celsius (8 C.).

(17) A vaccine containing the purified, inactivated virus is believed to be safer than the currently available attenuated, live YF virus vaccine because the disclosed inactivated YF virus vaccine is non-replicating. The inventors of the present subject matter have now developed a safer, inactivated, non-replicating YF vaccine that will elicit a neutralizing antibody response while eliminating the potential for neurotropic and viscerotropic adverse events. In addition, the improved vaccine can be manufactured by modern methods in Vero cells without animal derived proteins, and therefore it can be used safely in persons (including egg-allergic persons) for whom the live vaccine (produced in hens' eggs) is contraindicated or for whom warnings appear in the label. Such warnings would include, for example warnings to immunosuppressed persons, persons with thymic disease, egg-allergic persons, infants <9 months, and the elderly.

(18) Adaptation of Yellow Fever Virus for Robust Production in Vero Cells:

(19) The Vero cells used in the virus development phase were obtained from the World Health Organization (W.H.O.) seed lot, WHO Vero 10-87 Cell Bank at Passage 134. The WHO Vero 10-87 Cell Bank was originally made by the Institut Merieux using the ATCC Vero cell line CCL81 at Passage 129. The cells were thawed into OptiPRO SFM (serum-free medium) supplemented with 5% fetal bovine serum which was removed 24 hours later and replaced with OptiPRO SFM medium without fetal bovine serum. The serum, certified as being of USA origin, was gamma irradiated and had been tested for adventitious agents by the manufacturer; additional testing for sterility, mycoplasma, and adventitious viruses was performed on this material by WuXi AppTec. All subsequent passages of Vero cells to make the cell banks, virus seeds, and vaccine were made in OptiPRO SFM without serum. No other animal derived materials or products were used in producing the cell banks or the final vaccine according to an embodiment of the invention.

(20) Preparation of Vero Cell Banks:

(21) Master and Working Cell banks were prepared according to cGMP and were tested and characterized according to FDA Points to Consider. The Vero cells had an established provenance and were free from regulatory concerns about Bovine spongiform encephalitis (BSE). Serum-free growth medium was employed in propagating cells.

(22) Passage History of Vero Cells During Manufacture of Seed Viruses and Vaccine Lots:

(23) The passage history of Vero cells during the manufacture of the disclosed yellow fever vaccine is shown schematically in FIG. 1. The WHO cells were received at Passage 134, the Master Cell Bank (MCB) and Working Cell bank (MWCB) were banked at Passages 139 and 143 respectively. The cells were further expanded a maximum of 11 passages to Passage 154 during cell expansion in stationary cultures prior to seeding of the bioreactor used for virus production. The estimated number of population doublings in the bioreactor is calculated to be 1 to 3.

(24) Preparation of Master and Working Virus Seeds:

(25) FIG. 2 is a schematic representation of the preparation of Virus seeds according to an embodiment of the invention. An important safety factor for the disclosed vaccine is the use of the attenuated YF 17D vaccine for manufacture. The attenuated virus used as a starting material was a commercial vaccine, YF-VAX (Sanofi Pasteur, Swiftwater Pa.) which had undergone various tests for adventitious agents. The original YF-VAX material used to inoculate Vero cells was derived from embryonated hens' eggs, and contained hydrolyzed porcine gelatin as a stabilizer. However, the likelihood of carry-over of an adventitious agent from eggs was mitigated by use of RNA transfection to produce the Pre-Master Virus Seed.

(26) The cells were propagated in OptiPro-SFM medium (Invitrogen, Grand Island, N.Y.). To develop the modified Yellow Fever (YF) virus that will grow to high titers in Vero cells, initially the YF-17D virus at a 0.01 multiplicity of infection (MOI) was used to infect a T-25 flask with a confluent layer of Vero cells. The cell culture was incubated at 37 C. and 5 percent CO.sub.2.

(27) Once cytopathic effect (CPE) was observed in about 2+(50%) of the cells, aliquots of the culture were prepared, labeled as passage one (P1) and stored at 80 C. for use as the inoculum to continue the serial passages. A schematic of the procedure used to make P1 through P10 is shown in FIG. 3A.

(28) An aliquot of the Passage 1 (P1) virus was diluted 10.sup.1 through 10.sup.8 and each dilution was inoculated onto confluent monolayers of three (3) Vero cell cultures propagated in sterile 12 well plates from which growth medium had been removed.

(29) Log.sub.10 dilutions were prepared by transferring 0.2 ml of virus to 1.8 ml of phosphate buffered saline (PBS) to equal a 10.sup.1 dilution. The virus plus PBS was mixed and then a new pipette was used to transfer 0.2 ml to 1.8 ml of PBS=10.sup.2, and then repeated through 10.sup.8 dilution. Twelve well confluent monolayers of Vero cell culture were labeled and log.sub.10 dilutions of the P1 material (negative control, 10.sup.1 (3 wells), 10.sup.2 (3 wells), 10.sup.3 (3 wells), 10.sup.4 (3 wells), 10.sup.5 (3 wells), 10.sup.6 (3 wells), 10.sup.7 (3 wells) and 10.sup.8 (3 wells) were prepared and inoculated onto medium-free cultures using a new pipette for each dilution of inoculum. The negative control cultures were inoculated with a similar volume of PBS. After inoculating the cultures they were incubated at 37 C. for 1 hour with intermittent rocking and then 1.0 ml of maintenance medium was added per culture. Cells were observed each day for cytopathic effect (CPE) and recorded as 1+(25% of the cell monolayer effected), 2+(50% of the cell monolayer effected), 3+(75% of the cell monolayer effected) and 100% (all of the cell monolayer effected). Estimates of CPE were based on a comparison with the control cells. The plaque assay was also performed on the same dilutions of inoculum to verify that the CPE represented viral infectivity.

(30) Once CPE (2+) developed in these cultures, five 0.5 ml aliquots of the medium were harvested from the cultures that received the highest dilution or next to the highest dilution of inoculum. The five aliquots were prepared and stored as passage 2 (P2) at 80 C. The strategy was to select the virus population that replicated at or near the highest log.sub.10 dilution based on the appearance of CPE in the cells. As such, the virus population selected would be the population that was best adapted to replicate in the cells with possible genetic changes that will allow for an increase in viral titer.

(31) Subsequently, log.sub.10 dilutions were prepare of an aliquot of the P2 virus and used to infect cultures of Vero cell propagated in 12-well plates as described for passage one YF virus. Similar methods were employed to complete 10 serial passages of the virus.

(32) P10 and P11:

(33) At each serial passage, each of the aliquots used as the inoculum was also tested to determine the infectivity titers by plaque assay in Vero cells. At passage 10, five single, well isolated plaques, each representing progeny from a single infectious virus particle, were selected at the highest dilution that yielded plaques. Each plaque was suspended in 0.3 ml of medium containing Human Serum Albumin (HSA) to protect the virus infectivity during freezing and stored at 80 C.

(34) The series of passages (P1 to P10) of the YF 17D virus in Vero static cultures at dilutions of 10.sup.1 to 10.sup.8 were performed at the University of Texas Medical Branch (Galveston, Tex.). The strategy was to select the virus population that replicated at or near the highest log 10 dilution based on the microscopic appearance of CPE in the Vero cells. The virus population that showed cytopathic effects at the highest dilution, the P10 harvest, was selected as the optimized, high-yield virus. The high yield virus population that showed CPE at the highest dilution was sequenced.

(35) The High Yield Virus:

(36) The high yield virus was adapted for increased replication in Vero cells by 10 serial virus passages at terminal dilution in Vero cells. At Virus Passage 10, a single plaque forming unit was picked and passed in fluid culture to produce a mini-seed stock at Virus Passage 11. The graph in FIG. 3B shows comparative growth curves of P1 and P11 viruses, that had been inoculated at high MOI; the data indicate that the P11 virus has a higher peak titer than the P1 virus. This virus (P11) showed a 3-7 fold increased replication capacity in in Vero cells compared to the YF 17D at Virus Passage 1. The Virus Passage 11 virus stock was used for RNA extraction and the RNA used to produce cGMP grade virus seeds.

(37) RNA Sequence of the Vero Adapted 17D Virus (P11)

(38) The full genomic consensus sequences of the viruses at P1 and P11 from the original YF-VAX were determined Two genetic mutations or nucleotide differences were found, as shown in Table 1 below. One nucleotide difference lies in the capsid (C) gene and one in the envelope (E) gene. The term capsid as used herein, refers to the shell of protein that surrounds and protects the nucleic acid of a virus. The change in the C gene was silent (no amino acid change), whereas the E gene mutation resulted in an amino acid (Lys.fwdarw.Arg) mutation.

(39) TABLE-US-00001 TABLE 1 RNA sequence and mutations in the YF 17D virus adapted to Vero cells Nucleo- tide Amino Acid NT Change Change Codon residue # P1 P11 P1 P11 Location P1 P11 211 A G Threonine Threonine C31 ACA ACG 1452 A G Lysine Arginine E160 AAG AGG

(40) The first mutation was an A to G conversion at nucleotide residue #211, according to SEQ ID NO: 1, which resulted in a change in the codon for the amino acid at position 31 of the capsid protein (C31) from ACA to ACG. This mutation, however, did not change the amino acid residue at this position. The second mutation was an A to G conversion at nucleotide residue #1452, according to SEQ ID NO: 1, which resulted in a change in the codon for the amino acid at position 160 of the envelope protein (E160) from AAG to AGG. This mutation resulted in a Lysine to Arginine substitution at this position. A consensus alignment of the nucleic acid and amino acid sequences for P1 and P11 are depicted in FIGS. 4 and 5.

(41) Plaque Purification of P10 Harvest:

(42) As described above, virus from P10 was purified by plaque formation. The virus isolated from one plaque was inoculated into a T 150 flask. The conditioned medium from this flask was harvested when 50 percent of the cells exhibited CPE. This material was aliquoted in one mL aliquots and designated P11. The P11 virus was then used as the source of RNA for transfection of Vero cells. The P11 titer of plaque forming units was determined to be 8.510.sup.7 plaque forming units (PFU). The RNA isolated from the P 11 virus was used to transfect cells to produce a Pre-Master Seed. The Pre-Master Seed virus was passaged in additional cultures of Vero cells to produce a Master and Working Virus Seed stock.

(43) Manufacture of Master Virus Seed:

(44) The Master Virus Seed (MVS) was produced in Vero cells under serum-free conditions using a single vial of the Pre-Master Seed as the virus inoculum, as represented schematically in FIG. 2. Cells from the Manufacturer's Working Cell Bank (MWCB) of Vero cells at Passage 143 were expanded to eleven (11) 225 cm.sup.3 T-flasks. Once the cells became confluent, one flask was trypsinized and used to determine cell number and also to seed additional flasks used to produce the Working Virus Seed. The OptiPRO SFM medium was removed from the remaining 10 T-flasks and the cells were inoculated with Pre-Master Seed virus at a multiplicity of infection (MOI) 0.01 PFU/cell. The virus was allowed to adsorb for 60 (5) minutes at 372 C., after which pre-warmed OptiPRO SFM medium was added to the flasks. The infected culture was then incubated at 372 C. with 5% CO.sub.2.

(45) After 3 days, when CPE was observed in 80% of the cell population, the virus propagation process was terminated by harvesting the cell culture fluid. The virus-containing culture fluid was pooled from all flasks, centrifuged to remove cell debris, and mixed with sterile 70% sorbitol to a final sorbitol concentration of 7%. This mixture was filled into 4 mL cryovials at 2 mL per vial and frozen at 60 C. The frozen virus stock constitutes the YF 17D MVS.

(46) As shown in FIG. 2, the highest Vero cell passage level used for production of the MVS was 147.

(47) Manufacture of Working Virus Seed:

(48) The Working Virus Seed (WVS) was produced as shown in FIG. 2, from a single vial of the MVS under cGMP conditions. Starting with cells in the 11th T225 flask used to determine the cell density in the production of MVS, four T225 flasks were seeded at a cell density 110.sup.6 viable cells per flasks, Passage 147. The cells were passaged into 4 new T225 flasks to allow time for the production of the Master Seed Stock. Cells at Passage 148 were then seeded into eleven T225 flasks for the production of the WVS.

(49) When the cells were greater than 80% confluent, the cell density in one flask was determined. This cell density was used to estimate the cell density in the remaining ten flasks and the cells in the 10 flasks were infected with virus from the MVS at a MOI of 0.01 PFU/cell. To perform the infection, the medium was removed from the flasks and then diluted virus was added in phosphate buffered saline. After one hour fresh medium was added to each flask and the cells were returned to the incubator. The cells were observed microscopically for CPE. When CPE was greater than 80% the virus was harvested. The medium from the 10 flasks was centrifuged to remove cellular debris and the clarified supernatant was pooled into one vessel. Sorbitol (final concentration 7%) was added to the virus-containing supernatant as a cryo-preservative. The pooled virus was aliquoted into 4 mL cryovials, two mL per vial. The filled vials were stored at 60 C. Once frozen, one vial from the end of the bank was tested in a plaque assay in Vero cells to determine the virus titer.

(50) Increase in Titer Achieved in P11 Compared to P1:

(51) The original YF virus and P11 harvest of YF virus were titrated by plaque assay in Vero cells to determine the infectivity titers expressed as plaque forming units (PFU) (Table 1). The original YF-VAX 17D vaccine contained 10.sup.3.7 log.sub.10 per ml in Vero cells. The peak titer for passage one was 6.68 log.sub.10 per ml and remained at about the same titer through P6 and then increased significantly to 7.67 log.sub.10 by P10. Thus, in this experiment, there was a 1.0 log.sub.10 (10-fold) increase in the titer of the passage 10 (7.67 log.sub.10) over the titer (6.68 log.sub.10) of the P1 virus (see Table 2).

(52) Virus growth curves were also performed concurrently on the P1 and P11 viruses. Growth curves was performed by infecting duplicate 75 cm.sup.2 flasks of Vero cells at high MOI of 1.0 and a second growth curve was performed using a low MOI of 0.001. At high MOI it is expected that all cells are infected at initiation of the culture, while at low MOI, virus released by a small number of cells initially infected would infect the remaining cells of the culture; thus, virus in a low-MOI growth curve would be expected to be somewhat delayed compared to a high-MOI culture. At times 0, 6, 18, 24, 30, 48, 54 and 72 hr post inoculation, conditioned medium (2 mL) was removed from the cultures, stabilized with 2% HSA and frozen (duplicate one ml samples) at 80 C. Log.sub.10 dilutions of each sample were tested in Vero cells to determine the infectivity titer and the growth curves were plotted over time.

(53) TABLE-US-00002 TABLE 2 Peak infectivity titer for each sequential passage of YF virus Passages of YF- Highest Peak Conversion of plaque VAX in Dilutions Average infectivity forming units to equal the Vero yielding # of titer infectivity titer in cells plaques plaques (PFU/ml) log.sub.10 PFU per ml 0 10.sup.3 1 5 10.sup.3 3.7 1 10.sup.5 9.67 4.83 10.sup.6 6.68 2 10.sup.5 12.67 6.33 10.sup.6 6.80 3 10.sup.4 21.67 1.08 10.sup.6 6.03 4 10.sup.5 12.67 6.33 10.sup.6 6.80 6 10.sup.6 1.00 5.00 10.sup.6 6.70 8 10.sup.6 3.00 1.50 10.sup.7 7.18 9 10.sup.6 5.67 2.83 10.sup.7 7.45 10 10.sup.6 9.33 4.67 10.sup.7 7.67

(54) The growth curve results using an MOI of 1.0 indicated that the P1 YF virus increased from a titer of 4.09 log.sub.10 at 0 hours; or at the time of inoculation to a maximum titer of 6.28 log.sub.10 at 48 hours post inoculation (PI) and the titers showed a slight decrease of 6.21 and 6.18 log.sub.10 at 60 and 72 hours PI, respectively. The results for passage 11 (P11) showed an increase in titers over the passage one virus (P1). At the time of inoculation, the titer was 4.15 log.sub.10 and reached a maximum titer of 6.83 log.sub.10 at 48 hours P.I. and had decreased to a titer of 6.54 log.sub.10 at 72 hours P.I (see Table 3). The peak virus titer at approximately 48 hours for the P11 virus was 0.55 log.sub.10 or 3.5 times higher than for the P1 virus.

(55) TABLE-US-00003 TABLE 3 Growth curve of Yellow Fever 17D Passage 1 and Passage 11 virus at high MOI (1.0) Time points (hr) 0 6 18 24 30 48 54 72 Passage 1 4.15 4.11 5.63 6.09 6.05 6.28 6.21 6.18 Passage 11 4.09 4.22 5.60 6.27 6.63 6.83 6.68 6.54 P1 STDEV 0.11 0.02 0.17 0.08 0.05 0.03 0.05 0.04 P11 STDEV 0.04 0.08 0.21 0.02 0.10 0.14 0.18 0.10

(56) As compared to the growth curve using high MOI, the pattern of the growth curve using an MOI of 0.001 showed a lag in replication but maximum titers were higher. At the time of inoculation, the titers were 1.7 and 0.57 log.sub.10 for the passage 1 and 11, respectively. There was a linear increase in titers and by 72 hours PI, maximum titers of 7.35 and 8.17 log.sub.10 had been attained by P1 and P11, respectively. The peak virus titer at approximately 72 hours for the P11 virus was 0.82 log.sub.10 or 6.6 times higher than for the P1 virus. These results indicated that the serial passage of YF-VAX produced a substantial increase in titer and that this approach appears to be promising for developing an inactivated YF vaccine (see Table 4).

(57) TABLE-US-00004 TABLE 4 Growth curve of Yellow Fever 17D Passage 1 and Passage 11 virus at low MOI (0.001) Time points (hr) 0 6 18 24 30 48 54 72 Passage 1 1.70 2.00 2.57 4.14 4.72 6.44 7.19 7.35 Passage 11 0.57 0.67 3.01 4.44 5.18 7.04 7.38 8.17 P1 STDEV 0.00 0.30 0.19 0.07 0.06 0.04 0.02 0.03 P11 STDEV 0.98 1.15 0.14 0.05 0.05 0.02 0.10 0.10

(58) These results indicated that the serial passage of the YF virus produced a substantial increase in titer. Next, as described above, the sequence analysis of P1 and P11 was performed, the comparative results of which show that the serial passages may have resulted in two genetic mutations in the YF virus, one of which resulted in an amino acid change.

(59) The disclosed modified YF virus produced by the serial passage of the attenuated YF 17D virus vaccine in certified African green monkey kidney cells (Vero) showed enhanced productivity in cells. The methods of the invention involve vaccination of subjects with the modified, inactivated YF virus to produce immunity to Yellow Fever.

(60) Vaccine Production in Bioreactors:

(61) Bioreactors containing approximately 5 g/L of Cytodex 1 microcarriers were seeded with approximately 510.sup.5 Vero cells/mL in OptiPRO SFM medium. The cells were allowed to propagate for 3 to 4 days until cells attached to the microcarriers achieved a density of 710.sup.5 nuclei per mL. For virus inoculation, the agitation and parameter controls are turned off and the microcarriers and cells are allowed to settle. Approximately 75% of the medium volume was removed through a 90 m sieve tube which is designed to retain microcarriers in the reactor. WVS virus is introduced at a MOI of 0.01 PFU/cell. Low agitation was applied at this low volume for about 1 hour to allow virus to adsorb to and infect cells. Fresh medium was added to the full volume before agitation and parameter controls are returned to their original settings. On day 3 or 4 post infection, 75% of the conditioned medium was removed, and the reactor was re-fed with fresh medium. The culture was allowed to proceed for 2 or 3 more days and on Day 5, 6, or 7 post infection the conditioned medium was harvested. To ensure biosafety, harvest samples were taken from the bioreactor immediately before microcarrier removal and tested for sterility, mycoplasma, retroviruses and adventitious viruses (in vitro assay).

(62) The reactor mixing was stopped to allow for settling of the microcarriers. The culture is transferred from the bioreactor through a 90 m sieve tube into a bioprocess bag. The 90 m sieve reduces the amount of microcarriers and large particulates from transferring into the harvest. This was the Virus Harvest. The Virus Harvest was sampled and tested for infectivity, potency, identity, endotoxin, sterility, residual Vero cell DNA, and residual Vero cell proteins.

(63) Virus Purification and Inactivation:

(64) The culture conditioned medium was harvested, clarified in two steps, digested with BENZONASE, purified by ultrafiltration and diafiltration and then sterile filtered to generate the Live Virus Bulk. The Live Virus Bulk was then inactivated by treatment with -propiolactone (BPL) which permeates the virus envelope and disrupts the viral RNA by alkylating purine residues, rendering the virus inactive. The inactivated virus is further purified by cellufine sulfate column chromatography and diluted to the desired viral concentration to form the Bulk Vaccine Drug Substance.

(65) Repeat of YFV 17D Passaging Study:

(66) Experiments were performed to repeat the passage of YF virus from unpassaged virus stock through P11 using similar techniques as in the original passage series.

(67) Preparation of the Virus Stocks:

(68) Vero cells were maintained under serum-free conditions throughout the study, using OptiPRO SFM.

(69) The initial source of the YFV 17D virus was from a single vial of YF-VAX (Sanofi Pasteur, Swiftwater Pa.). The vial was originally reconstituted and dispensed into aliquots. One of these aliquots was used for the repeat experiments. The repeat serial passaging was performed in duplicate such that there were two runs of the study, performed in parallel, referred to here as series B and C.

(70) At each passage of the virus, the virus sample was diluted in serial 10-fold dilutions, and the diluted virus was used to inoculate Vero cells seeded in 12 well plates. The serial dilutions performed at each passage were inoculated in duplicate such that one set of plates was used for the preparing the next passage of virus, inoculating 4 wells per dilution, and the other set of plates was used to determine the titer of the passaged virus, inoculating 2 wells per dilution.

(71) For the serial passages of the virus, the dilution selected for passaging the virus was the last dilution where generalized cytopathic effect (CPE) was observed, three to four days after infection. The media from the four wells was pooled for the next passage. The titer of the virus was determined by plaque assay using an immunostain to visualize and count the plaques. The immunostain method allowed for determining the titer after 3 days of infection.

(72) For the initial passage of the virus, 0.3 ml of the YF-VAX aliquot was diluted into 3 mL final, using OptiPRO SFM, for a 10.sup.1 dilution. The diluted virus was divided equally into three aliquots. From each of these aliquots, serial 10-fold dilutions were made to 10.sup.5, making two dilution series (B and C). This is referred to here as the P0.fwdarw.P1 passage. From the plaque assay inoculated using the dilution series, of the P0 virus was determined, and for the plates inoculated for passage, the P1 virus was generated. Each round of the passaging is summarized in the Table 5.

(73) TABLE-US-00005 TABLE 5 Serial Passages of YFV 17D (Results same for series B and C) Dilution harvested Passage Dilutions plated for next passage P0 (initial vial) N/A N/A P0 .fwdarw. P1 10.sup.1 to 10.sup.5 10.sup.3 P1 .fwdarw. P2 10.sup.2 to 10.sup.7 10.sup.5 P2 .fwdarw. P3 10.sup.3 to 10.sup.8 10.sup.5 P3 .fwdarw.P4 10.sup.3 to 10.sup.9 10.sup.5 P4 .fwdarw.P5 10.sup.3 to 10.sup.9 10.sup.5 P5 .fwdarw.P6 10.sup.3 to 10.sup.9 10.sup.4 P6 .fwdarw.P7 10.sup.3 to 10.sup.9 10.sup.5 P7 .fwdarw.P8 10.sup.3 to 10.sup.7 10.sup.4 P8.fwdarw.P9 10.sup.3 to 10.sup.7 10.sup.5 P9 .fwdarw.P10 10.sup.3 to 10.sup.7 10.sup.5

(74) The passaging was repeated for 10 serial passages of the virus. Once the virus was harvested from the last passage, the titers were generated for the P10 virus from each series. The P10 viruses were then diluted for inoculating cells such that only one plaque per well would develop after inoculation. Well-isolated plaques could then be picked from the wells. From the B series, six well-isolated plaques were picked, and from C, two were picked. The picked plaques were used to inoculate T25 flasks to generate the P11 virus stocks for growth curve studies.

(75) Growth Curve Analysis:

(76) For the growth curve studies, the P1 stock virus from each series was compared to the P11 stocks for each series. Since the volume of the P1 stocks would have been limiting for this, an aliquot of P1 virus from each series was diluted three-fold, then aliquots made from the diluted virus to generate P1 stocks for the growth curve. For the P11 stocks, three stocks from the B series were analyzed, and the two from the C series. Prior to the growth curve studies, aliquots of the virus stocks were assayed to confirm the level of infectivity.

(77) The growth curve analysis was performed by infecting Vero cells in T25 flasks, at low MOI of 0.001 PFU/cell. The study was conducted under serum free conditions using OptiPRO SFM as the culture medium. After diluting the virus stocks to achieve the target 0.001 MOI, a sample was reserved to confirm the titer of the inoculum. The virus inocula were allowed to adsorb to the cells for approximately one hour. After adsorption, the monolayers were washed three times then the cultures were fed with 8 ml of medium. At each time point, 1 mL was removed from each culture, and 1 mL fresh media was added back. The reserved one mL of medium was clarified by centrifugation and stored at 80 C. in the presence of sorbitol, until ready to assay. The time points for which samples were taken were 0, 24, 30, 48, 54, 72, and 81 hours after infection. A plaque assay was performed on all samples. The results of the study are detailed in FIG. 3C.

(78) For both the B and C series, there was one P11 virus stock that was shown to replicate to higher titers than the P1 virus stock from the series. The P1 stocks for both B and C, and the B3-P11 stock and the C1-P11 stock were selected for sequence analysis. The sequence analysis illustrates that the B3 stock enjoys the same Lys.fwdarw.Arg mutation at E160 as was observed in the original passage series, as well as a Threonine (Thr).fwdarw.Isoleucine (Ile) mutation at amino acid position 317 in non-structural protein 1 (NS1-317), and a Phenylalanine (Phe).fwdarw.Leucine (Leu) mutation at amino acid position 170 in non-structural protein 2A (NS2A-170). While, the C1 stock did not carry the same mutation in the E gene, further study of the C1 stock genome is ongoing, and has revealed a mutation at amino acid position 113 in non-structural protein NS4B (NS4B-113).

(79) Table 6 summarizes the nucleotide and amino acid changes found in the modified Yellow Fever viruses obtained from the original and repeat passage studies.

(80) TABLE-US-00006 TABLE 6 Nucleotide and amino acid changes in the consensus sequence between passage 1 (P1) and P11 in three separate passage series of yellow fever 17D vaccine (YF-VAX) in Vero cells. The position of the altered nucleotide or amino acid in the designated viral protein (or non-coding region, NCR) is shown. Some nucleotide changes were silent (did not result in corresponding amino acid mutations). Original passage series Repeat Series B Repeat Series C Amino Amino Amino Protein Nucleotide acid Nucleotide acid Nucleotide acid 5NCR prM E 211 A.fwdarw.G 211 A.fwdarw.G 1452 A.fwdarw.G 160 K.fwdarw.R 1452 A.fwdarw.G 160 K.fwdarw.R 1507 T.fwdarw.C 1897 G.fwdarw.A NS1 3402 C.fwdarw.T 317 T.fwdarw.I NS2a 4016 T.fwdarw.C 170 F.fwdarw.L NS2b NS3 NS4a NS4b 7225 A.fwdarw.G 113 I.fwdarw.M NS5 3NCR 9343 G.fwdarw.A 9670 C.fwdarw.T
Non-Limiting Aspects of the Invention:

(81) A Yellow Fever viral strain was produced to develop a safer, inactivated, non-replicating vaccine that will elicit a neutralizing antibody response while eliminating the potential for neurotropic and viscerotropic adverse events for the prevention of human disease. Additional Yellow Fever virus strains are produced to develop safer, inactivated, non-replicating vaccines that will elicit a neutralizing antibody response while eliminating the potential for neurotropic and viscerotropic adverse events for the prevention of human disease. These embodiments of the invention are set forth above in the Summary.

(82) The invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises an amino acid mutation at one or more positions flanking the 160 mutation, for example residues 134, 137, 144, 148, 157, 160, 175, 177 of the envelope protein. In an embodiment of this aspect, the invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises at least one amino acid mutation selected from: an amino acid mutation in the NS1 protein, an amino acid mutation in the NS2A protein, an amino acid mutation in the NS4B protein, optionally wherein said at least one amino acid mutation is in further combination with an amino acid mutation at one or more positions 134, 137, 144, 148, 157, 160, 175, 177 of the envelope protein. In a further embodiment of this aspect, the amino acid mutation(s) at position 157 is lysine to arginine; at position 148 is lysine to arginine; at position 144 is histidine to arginine, tyrosine or lysine; at position 137 is tyrosine to arginine or lysine, at position 175 is tyrosine to arginine or lysine; and/or at position 177 is lysine to arginine.

(83) The invention also provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises an amino acid mutation at one or more positions flanking the 160 mutation, for example residues 134, 137, 144, 148, 157, 160, 175, 177 of the envelope protein, in combination with mutations at one or more positions 317 of NS1, 170 of NS2A, 113 of NS4B. In an embodiment of this aspect, the amino acid mutation(s) in the envelope protein at position 157 is lysine to arginine; at position 148 is lysine to arginine; at position 144 is histidine to arginine, tyrosine or lysine; at position 137 is tyrosine to arginine or lysine; at position 175 is tyrosine to arginine or lysine; and/or at position 177 is lysine to arginine; and the amino acid mutation in NS1 at position 317 is threonine to isoleucine, in NS2A at position 170 is phenylalanine to leucine, in NS4B at position 113 is isoleucine to methionine.

(84) In embodiments according to certain aspects of the invention, the cells are selected from Vero cells. Other cells suitable for propagation of the Yellow Fever virus may utilized, including but not limited to, primary chick embryo, primary duck embryo, primary dog kidney, primary rabbit kidney, WI-38, MRC-5, or fetal rhesus lung.

(85) In some embodiments of these aspects, the nucleotide mutation in the codon for the amino acid at position 160 of the envelope protein results in a change from AAG to AGG, AGA, CGC, CGA, CGG or CGU. In other embodiments of these aspects, the amino acid mutation at position 160 is lysine to arginine.

(86) In still other embodiments of these aspects, the nucleotide mutation in the codon for the amino acid at position 317 of NS1 results in a change from ACA to AUA, the nucleotide mutation in the codon for the amino acid at position 170 of NS2A results in a change from UUU to CUU, the nucleotide mutation in the codon for the amino acid at position 113 of NS4B results in a change from AUA to AUG. In other embodiments of these aspects, the amino acid mutation at position 317 of NS1 is threonine to isoleucine, at position 170 of NS2A is phenylalanine to leucine, at position 113 of NS4B is isoleucine to methionine.

(87) In the methods according to the various aspects of the invention, the Yellow Fever virus or vaccines of the invention can be administered in amounts and by using methods that can readily be determined by persons of ordinary skill in this art. The chemically inactivated viral vaccines can be administered and formulated, for example, as a sterile aqueous solution containing between 10.sup.2 and 10.sup.8, e.g., or between 10.sup.6 and 10.sup.7, inactivated equivalents of infectious units (e.g., plaque-forming units (PFU) or tissue culture infectious doses) in a dose volume of from about 0.1 to about 1.0 ml, or about 0.5 ml. to be administered by, for example, subcutaneous, intramuscular, epidermal, or intradermal routes. In addition, in an appropriate formulation, a mucosal route, such as the intranasal oral route, can be selected. Selection of an appropriate amount of virus to administer can be determined by those of skill in this art, and this amount can vary due to numerous factors, e.g., the size and general health of the subject to whom the virus is to be administered. The subject can be vaccinated a single time or, if necessary, follow-up immunization can take place.

(88) As is noted above, the vaccines can be administered as primary prophylactic agents to a subject that is at risk of Yellow Fever virus infection. Also, although not required, adjuvants can be used to enhance the immunogenicity of the Yellow Fever virus vaccines. Selection of appropriate adjuvants can readily be carried out by those of skill in this art.

(89) Also as is noted above, the live virus can be inactivated by treatment with -propiolactone (BPL), rendering the virus inactive. Other suitable methods of virus inactivation include, but are not limited to, formalin, ultraviolet radiation, ethylenimine, acetylethylenimine, and binary ethylenimine

EXEMPLIFICATION

(90) The examples below are intended to further illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention.

(91) Antibody Responses in Mice:

(92) The neutralizing antibody responses in female, outbred BALB/c and CD-1 mice after immunization with inactivated yellow fever vaccine compared to live virus was assessed. Yellow fever (YF) virus was inactivated with beta propiolactone (BPL), formulated with alum adjuvant and injected by the intramuscular route as two or three doses, each separated by 14 days. Two dose levels of virus were tested in BALB/c mice, the high dose level only was tested in CD1 mice. Sera taken at 14 days after the last immunization were tested for neutralizing antibody activity.

(93) Preimmunization Procedures:

(94) Female BALB/c and CD-1 strain mice (6 weeks of age) were acclimated in designated isolators in a restricted virus animal facility. Serum sample were collected Study Day 28 or 42 upon sacrifice. Mice were housed at 5 mice per cage and each animal was uniquely identified on the cage cards, and by ear notch. Mice were acclimated for a week prior to the initiation of any treatments. Mice received sterilized food and water and were housed in sterilized polycarbonate cages with sterilized bedding with a 12-hour light cycle (on at 6 am and off at 6 pm). General health was evaluated by technical staff daily and by a veterinarian weekly and as needed for health issues. Body weights were collected on Day 0 prior to immunization and on Day 28 and 42.

(95) Immunization Procedure:

(96) Body weight was determined on Day 0 prior to immunization Immunization was given by either the i.m. (alum formulations) or s.c. (live virus or inactivated vaccine with Freund's adjuvant) route. Injections were given with mice under light anesthesia with suboptimal dose of ketamine/xylazine mixture. For s.c. route with live virus, a volume of 100 l of vaccine in a 1 ml syringe fitted with a 27 gauge needle is injected between the skin and underlying layers of tissue in the scapular region on the backs of mice. For i.m. administration, a volume of 100 l of vaccine in a 0.5 ml insulin syringe is injected into the muscle bundles of 2 rear upper legs of mice (50 l/leg).

(97) Sacrifice:

(98) Mice were sacrificed 28 or 42 days after the first vaccination. Body weight was determined on all mice on Study Day 28 and prior to sacrifice. Blood was collected for neutralizing antibody testing. Blood (0.7-1.0 ml) was removed by cardiac puncture from mice anesthetized with light ketamine/xylazine treatment before they are humanely terminated by ketamine/xylazine overdose.

(99) Experimental Design:

(100) Alum-formulated vaccine prepared the day prior to immunization as a suspension and the vaccine was well mixed prior to filling each syringe. Alum-formulated preparations were administered by the i.m. route, a volume of 100 l of vaccine in a 0.5 ml insulin syringe was injected into the muscle bundles of 2 rear upper legs of mice (50 l/leg).

(101) Live Yellow Fever (YF) vaccine was reconstituted with 0.6 ml of saline to a virus concentration of approximately 1.110.sup.5 pfu/ml. A dose of 110.sup.4 PFU (i.e. 1/10.sup.th the human dose) was delivered in a volume of 100 l of sterile saline administered on day 0 s.c.

(102) Freund's adjuvanted vaccine was formulated the day of vaccination by placing 2 ml of antigen solution into a glass syringe, and 2 ml of the adjuvant into another glass syringe. The syringes were connected through the luer fitting to the 3-way valve. The plunger from the antigen solution was carefully depressed first, pushing the antigen into the oil of the adjuvant. The plungers were alternately pushed, to mix the adjuvant and the antigen solution into an emulsion (approximately 8 to 10 minutes). A 0.5 ml volume was delivered s.c. between the skin and underlying layers of tissue in the scapular region on the backs of mice (Formulation with Freund's adjuvant).

(103) Live Yellow Fever (YF Vax) vaccine was reconstituted with 0.6 ml of supplied saline to a virus concentration of approximately 1.110.sup.5 PFU/ml.

(104) The inactivated whole virion vaccine adsorbed to 0.2% aluminum hydroxide (alum) adjuvant was prepared no more than 2 weeks prior to day of dosing.

(105) Preliminary Mouse Studies

(106) Groups of 5 mice each were dosed with as outlined in Table 7. Serum samples were collected by cardiac puncture 14 or 28 days post last vaccination.

(107) TABLE-US-00007 TABLE 7 Vaccination Group # Mice Strain Vaccine (Volume = 0.1 ml) Route schedule Neut. Ab 1 5 BALB/c 10.sup.8 BPL-inactivated in 0.2% IM Day 0, 14 Day 28 alum 2 5 BALB/c 10.sup.8 BPL-inactivated in 0.2% IM Day 0, 14, 28 Day 42 alum 3 5 BALB/c 10.sup.7 BPL-inactivated in 0.2% IM Day 0, 14 Day 28 alum 4 5 BALB/c 10.sup.7 BPL-inactivated in 0.2% IM Day 0, 14, 28 Day 42 alum 5 5 BALB/c 10.sup.8 BPL-inactivated in SC Day 0, 14, 28 Day 42 Freund's complete/incomplete 6 5 BALB/c 10.sup.7 BPL-inactivated Freund's SC Day 0, 14, 28 Day 42 complete/incomplete 7 5 BALB/c 10.sup.8 BPL-inactivated no IM Day 0, 14, 28 Day 42 adjuvant 8 5 BALB/c Live YF Vax SC Day 0 Day 28 9 5 CD1 10.sup.8 BPL-inactivated in 0.2% IM Day 0, 14 Day 28 alum 10 5 CD1 10.sup.8 BPL-inactivated in 0.2% IM Day 0, 14, 28 Day 42 alum 11 5 BALB/c 0.2% alum IM Day 0, 14, 28 Day 42
Plaque Reduction Neutralization Activity in Mouse Sera

(108) Plaque reduction neutralization test was performed using a dilution of 17D virus which, in the absence of neutralization, produces 10-40 plaque forming units per well in 12 well plates. An equal volume of serially diluted mouse serum was incubated with virus for 16-20 h at 4 C. and then the inoculated into duplicate wells of Vero cells in 12 well plates. After virus absorption for 60 minutes at 37 C., the wells are overlaid with medium containing 0.75% methylcellulose, incubated for 4 days at 37 C., fixed and stained with crystal violet and plaques counted using a stereomicroscope over light box. The 50% plaque reduction titer represents the final mouse serum dilution resulting in less than 50% of the average plaque counts when no serum is added.

(109) The plaque reduction neutralization test (PRNT) responses and titers are shown in Table 8 and FIGS. 6 and 7. The PRNT test is currently the generally accepted standard for antibodies against Yellow Fever virus. All mice, regardless of strain, receiving 2 or 3 doses of inactivated vaccine given either without adjuvant (Group 7), with alum (Groups 1, 2, 3, 4, 9, 10, 11), or with Freund's adjuvant (Groups 5, 6) developed neutralizing antibody responses. Titers of greater than 4096 were found in 5 of 5 BALB/c mice immunized with 3 doses of alum bound inactivated virus at the 10.sup.7 EU/dose. These titers were higher than BALB/c mice immunized with 3 doses of inactivated virus delivered with Freund's adjuvant (Group 6, titers 16-128). CD1 mice immunized with 2 doses of alum bound inactivated virus at the 10.sup.8 EU/dose level achieved higher titers (Group 9; titers 512-1024) than did similarly immunized BALB/c mice (Group 3; titers of 32-64). Only 1 in 5 mice receiving live YF Vax (Group 8) mounted a neutralizing antibody response that was above the baseline levels in the mice receiving alum only (Groups 11).

(110) In FIG. 7, each symbol represents an individual mouse. Treatment groups are shown in Tables 7 and 8. For Group 6 (*) the highest dilution of serum tested was 1:128. For Group 9 (**) the highest dilution of serum tested was 1:2048.

(111) This study demonstrates that robust neutralizing antibody titers can be achieved in mice immunized with 2 or more inoculations of the disclosed inactivated YF virus delivered with alum. Outbred CD1 mice had higher antibody responses than an inbred strain (BALB/c). Alum was a superior adjuvant to Freund's, but this result could also be related to the route of immunization (SC for Freund's vs. IM for alum). Additional studies will be performed to determine if immunogenicity can be achieved with a single dose of vaccine.

(112) TABLE-US-00008 TABLE 8 Mice with plaque reduction neutralization activity Schedule % Strain Vaccina- Positive Group of mice Vaccine tion Sacrifice (+/total) 1 BALB/c 10.sup.8 BPL-inactivated Day 0, 14 Day 28 100% in 0.2% alum (5/5) 2 BALB/c 10.sup.8 BPL-inactivated Day 0, 14, Day 42 100% in 0.2% alum 28 (5/5) 3 BALB/c 10.sup.7 BPL-inactivated Day 0, 14 Day 28 100% in 0.2% alum (5/5) 4 BALB/c 10.sup.7 BPL-inactivated Day 0, 14, Day 42 100% in 0.2% alum 28 (5/5) 5 BALB/c 10.sup.8 BPL-inactivated Day 0, 14, Day 42 100% in Freund's 28 (5/5) complete/incomplete 6 BALB/c 10.sup.7 BPL-inactivated Day 0, 14, Day 42 100% in Freund's 28 (5/5) complete/incomplete 7 BALB/c 10.sup.8 BPL-inactivated Day 0, 14, Day 42 100% no adjuvant 28 (5/5) 8 BALB/c Live YF Vax Day 0 Day 28 20% (1/5) 9 CD1 10.sup.8 BPL-inactivated Day 0, 14 Day 28 100% in 0.2% alum (5/5) 10 CD1 10.sup.8 BPL-inactivated Day 0, 14, Day 42 100% in 0.2% alum 28 (5/5) 11 BALB/c 0.2% alum Day 0, 14, Day 42 0% 28 (0/5)

EQUIVALENTS

(113) While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. Additionally, the references, patents and patent publications cited herein are incorporated by reference in their entirety.