DEOPTIMIZED YELLOW FEVER VIRUS AND METHODS AND USES THEREOF

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronic xml file named “SequenceListing_064955_000050W000_ST26”, having a size in bytes of 70,255 bytes, and created on Jul. 5, 2022. The information contained in this electronic file is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to deoptimized Yellow Fever virus vaccine strain and methods of using deoptimized Yellow Fever virus vaccine strain, as immune compositions and to induce oncolytic effects on malignant tumors and to treat malignant tumors.

BACKGROUND

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Yellow Fever Virus

Tropical diseases of viral etiology such as Yellow Fever (YF) pose significant health risks to many people in the world, including service members as the US continues to deploy troops overseas. YF has a high mortality and lacks a specific therapy.

All current YF vaccines are based on the 17D live attenuated strain of YF virus. Although highly effective, the 17D vaccines are frequently reactogenic, with side effects including headache, myalgia, low grade fever and chills for 5-10 days following vaccination. In addition, very rare but serious vaccine-related adverse events and deaths have been documented. Since current 17D vaccines must be manufactured in chicken embryos and cannot be purified to modern standards, some of the observed reactogenicity may be a result of the high levels of egg and chicken embryo derived by-products in the vaccines. Furthermore, owing to their nature as biological isolates and their long passage history, 17D vaccine seeds are genetically poorly defined swarms of quasispecies mutants. Some more virulent mutants present in the vaccines at a low frequency may have an outsized effect on the reactogenicity of the vaccine.

The development of a new YF vaccine that is safer, less reactogenic, and manufacturable in modern cell culture systems is vital to provide protection to the global population.

Synthetic Virology

Rapid improvements in DNA synthesis technology promise to revolutionize traditional methods employed in virology. One of the approaches traditionally used to eliminate the functions of different regions of the viral genome makes extensive but laborious use of site-directed mutagenesis to explore the impact of small sequence variations in the genomes of virus strains. However, viral genomes, especially of RNA viruses, are relatively short, often less than 10,000 bases long, making them amenable to whole genome synthesis using currently available technology. Recently developed microfluidic chip-based technologies can perform de novo synthesis of new genomes designed to specification for only a few hundred dollars each. This permits the generation of entirely novel coding sequences or the modulation of existing sequences to a degree practically impossible with traditional cloning methods. Such freedom of design provides tremendous power to perform large-scale redesign of DNA/RNA coding sequences to: (1) study the impact of changes in parameters such as codon bias, codon-pair bias, and RNA secondary structure on viral translation and replication efficiency; (2) perform efficient full genome scans for unknown regulatory elements and other signals necessary for successful viral reproduction; (3) develop new biotechnologies for genetic engineering of viral strains and design of anti-viral vaccines; (4) synthesize deoptimized viruses for use in oncolytic therapy.

De Novo Synthesis of Viral Genomes

Computer-based algorithms are used to design and synthesize viral genomes de novo. These synthesized genomes, exemplified by the synthesis of Yellow Fever virus 17D, can be used to treat cancer.

It has been known that malignant tumors result from the uncontrolled growth of cells in an organ. The tumors grow to an extent where normal organ function may be critically impaired by tumor invasion, replacement of functioning tissue, competition for essential resources and, frequently, metastatic spread to secondary sites. Malignant cancer is the second leading cause of mortality in the United States.

Prior art methods for treating malignant tumors include surgical resection, radiation and/or chemotherapy. However, numerous malignancies respond poorly to all traditionally available treatment options and there are serious adverse side effects to the known and practiced methods. There has been much advancement to reduce the severity of the side effects while increasing the efficiency of commonly practiced treatment regimens. However, many problems remain, and there is a need to search for alternative modalities of treatment.

In recent years, there have been proposals to use viruses for the treatment of cancer: (1) as gene delivery vehicles; (2) as direct oncolytic agents by using viruses that have been genetically modified to lose their pathogenic features; or (3) as agents to selectively damage malignant cells using viruses which have been genetic engineered for this purpose.

Examples for the use of viruses against malignant gliomas include the following. Herpes Simplex Virus dlsptk (HSVdlsptk), is a thymidine kinase (TK)-negative mutant of HSV. This virus is attenuated for neurovirulence because of a 360-base-pair deletion in the TK gene, the product of which is necessary for normal viral replication. It has been found that HSVdlsptk retains propagation potential in rapidly dividing malignant cells, causing cell lysis and death. Unfortunately, all defective herpes viruses with attenuated neuropathogenicity have been linked with serious symptoms of encephalitis in experimental animals. For example, in mice infected intracerebrally with HSVdlsptk, the LD₅₀Ic (intracranial administration) is 10⁶pfu, a rather low dose. This limits the use of this mutant HSV. Other mutants of HSV have been proposed and tested. Nevertheless, death from viral encephalitis remains a problem.

Another proposal was to use retroviruses engineered to contain the HSV tk gene to express thymidine kinase which causes in vivo phosphorylation of nucleoside analogs, such as gancyclovir or acyclovir, blocking the replication of DNA and selectively killing the dividing cell. Izquierdo, M., et al., Gene Therapy, 2:66-69 (1995) reported the use of Moloney Murine Leukemia Virus (MoMLV) engineered with an insertion of the HSV tk gene with its own promoter. Follow-up of patients with glioblastomas that were treated with intraneoplastic inoculations of therapeutic retroviruses by MRI revealed shrinkage of tumors with no apparent short-term side effects. However, the experimental therapy had no effect on short-term or long-term survival of affected patients. Retroviral therapy is typically associated with the danger of serious long-term side effects (e.g., insertional mutagenesis).

Similar systems have been developed to target malignancies of the upper airways, tumors that originate within the tissue naturally susceptible to adenovirus infection and that are easily accessible. However, Glioblastoma multiforme, highly malignant tumors composed of widely heterogeneous cell types (hence the denomination multiforme) are characterized by exceedingly variable genotypes and are unlikely to respond to oncolytic virus systems directed against homogeneous tumors with uniform genetic abnormalities.

The effects of the virus modification described herein can be confirmed in ways that are known to one of ordinary skill in the art. Non-limiting examples induce plaque assays, growth measurements, reverse genetics of RNA viruses, and reduced lethality in test animals. The instant application demonstrates that the deoptimized viruses are capable of inducing protective immune responses in a host as well as inducing an anti-tumor response in the host.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

Various embodiments of the invention provide for a polynucleotide comprising a polynucleotide encoding one or more viral proteins or one or more fragments thereof of a parent Yellow Fever virus (YFV): wherein the polynucleotide is recoded compared to its parent YFV, wherein the amino acid sequence of the one or more viral proteins, or the one or more fragments thereof of the parent YFV encoded by the polynucleotide remains the same, or wherein the amino acid sequence of the one or more viral proteins or the one or more fragments thereof of the parent YFV encoded by the polynucleotide comprises one or more amino acid substitutions, additions, or deletions.

In various embodiments, the one or more viral proteins or one or more fragments thereof can be the E protein or a fragment thereof.

In various embodiments, the E protein or a fragment thereof can be encoded by a polynucleotide having SEQ ID NOs:7, 3, 9, 8, 4, 5 or 6, or a fragment thereof, or a variant of a polynucleotide having SEQ ID NOs:7, 3, 9, 8, 4, 5 or 6, or a fragment thereof. In various embodiments, the E protein or a fragment thereof can be encoded by a polynucleotide having SEQ ID NO:7. In various embodiments, the E protein or a fragment thereof can be encoded by a polynucleotide having SEQ ID NO:3. In various embodiments, the E protein or a fragment thereof can be encoded by a polynucleotide having SEQ ID NO:9. In various embodiments, the E protein or a fragment thereof can be encoded by a polynucleotide having SEQ ID NO:8. In various embodiments, the polynucleotide sequence can have SEQ ID NOs:7, 3, 9, 8, 4, 5 or 6.

In various embodiments, the parent YFV can be YFV strain 17D (YFV 17D), or has at least 95%, 96%, 97%, 98%, or 99% sequence identity to YFV 17D.

In various embodiments, the parent YFV can be YFV 17D-204, YFV 17DD, or YFV 17D-213, or has at least 95%, 96%, 97%, 98%, or 99% sequence identity to YFV 17D-204, YFV 17DD, or YFV 17D-213.

Various embodiments provide for a polypeptide encoded by any one of the polynucleotides of present invention as discussed herein.

Various embodiments provide for a deoptimized Yellow Fever virus (YFV) comprising any one of the polynucleotides of present invention as discussed herein.

Various embodiments provide for a deoptimized Yellow Fever virus (YFV) comprising a polypeptide encoded by any one of the polynucleotides of present invention as discussed herein.

Various embodiments provide for a deoptimized Yellow Fever virus (YFV) of the present invention, wherein expression of one or more of its viral proteins is reduced compared to its parent YFV.

Various embodiments provide for an immune composition or vaccine composition comprising a deoptimized YFV the present invention as discussed herein.

Various embodiments provide for a method of treating a malignant tumor or reducing tumor size, comprising: administering a deoptimized Yellow Fever virus (YFV) of the present invention as discussed herein or an immune composition of the present invention as discussed herein to a subject in need thereof.

Various embodiments provide for a method of treating a malignant tumor, comprising: administering a prime dose of deoptimized YFV of the present invention as discussed herein, or an immune composition of the present invention as discussed herein, to a subject in need thereof, and administering one or more boost dose of deoptimized YFV of the present invention as discussed herein, or an immune composition of the present invention as discussed herein, to the subject in need thereof.

Various embodiments provide for a method of reducing tumor size, comprising administering a prime dose of a deoptimized YFV of the present invention as discussed herein, or an immune composition of the present invention as discussed herein, to a subject in need thereof, and administering one or more boost dose of a deoptimized YFV of the present invention as discussed herein, or an immune composition of the present invention as discussed herein, to the subject in need thereof.

In various embodiments, the deoptimized YFV can be deoptimized YFV strain 17D (YFV 17D).

In various embodiments, the deoptimized YFV can be deoptimized YFV 17D-204, deoptimized YFV 17DD, or deoptimized YFV 17D-213.

In various embodiments, the prime dose can be administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously.

In various embodiments, the one or more boost dose can be administered intratumorally or intravenously.

In various embodiments, a first of the one or more boost dose can be administered about 2 weeks after one prime dose, or if more than one prime dose then about 2 weeks after the last prime dose.

In various embodiments, the subject can have cancer.

In various embodiments, the prime dose can be administered when the subject does not have cancer.

In various embodiments, the subject can be at a higher risk of developing cancer.

In various embodiments, the one or more boost dose can be administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years after the prime dose when the subject does not have cancer.

In various embodiments, the subject can be subsequently diagnosed with cancer and the one or more boost dose can be administered after the subject is diagnosed with cancer.

In various embodiments, the method can further comprise administering a PD-1 inhibitor or a PD-L1 inhibitor. In various embodiments, the PD-1 inhibitor can be an anti-PD1 antibody. In various embodiments, the anti-PD1 antibody can be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, AK105, BCD-100, BI 754091, JS001, LZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb20717, tislelizumab, and combinations thereof. In various embodiments, the PD-1 inhibitor can be selected from the group consisting of PF-06801591, anti-PD1 antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In various embodiments, the PD-L1 inhibitor can be an anti-PD-L1 antibody. In various embodiments, the anti-PD-L1 antibody can be selected from the group consisting of BGB-A333, CK-301, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316, atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, and combinations thereof. In various embodiments, the anti-PD-L1 inhibitor can be M7824.

In various embodiments, treating the malignant tumor can decrease the likelihood of recurrence of the malignant tumor. In various embodiments, treating the malignant tumor can decrease the likelihood of having a second cancer that is different from the malignant tumor.

In various embodiments, if the subject develops a second cancer that is different from the malignant tumor, the treatment of the malignant tumor can result in slowing the growth of the second cancer.

In various embodiments, after remission of the malignant tumor, if the subject develops a second cancer that is different from the malignant tumor, the treatment of the malignant tumor can result in in slowing the growth of the second cancer.

In various embodiments, treating the malignant tumor can stimulate an inflammatory immune response in the tumor. In various embodiments, treating the malignant tumor can recruit pro-inflammatory cells to the tumor. In various embodiments, treating the malignant tumor can stimulate an anti-tumor immune response.

In various embodiments, the malignant tumor can be a solid tumor. In various embodiments, the malignant tumor can be selected from a group consisting of glioma, neuroblastoma, glioblastoma multiforme, adenocarcinoma, medulloblastoma, mammary carcinoma, prostate carcinoma, colorectal carcinoma, hepatocellular carcinoma, bladder cancer, prostate cancer, lung carcinoma, bronchial carcinoma, epidermoid carcinoma, and melanoma.

In various embodiments, the deoptimized YFV can be administered intratumorally, intravenously, intracerebrally, intramuscularly, intraspinally or intrathecally.

In various embodiments, administering the deoptimized YFV can cause cell lysis in the tumor cells.

Various embodiments provide for a method of eliciting an immune response in a subject in need thereof, comprising administering a deoptimized Yellow Fever virus (YFV) of the present invention, or an immune or vaccine composition of the present invention, to a subject in need thereof. In various embodiments, the method elicits an immune response against YFV.

Various embodiments provide for a method of eliciting an immune response in a subject, comprising: administering a prime dose of deoptimized YFV of the present invention, or an immune or vaccine composition of the present invention, to a subject in need thereof, and administering one or more boost dose of deoptimized YFV of the present invention, or an immune or vaccine composition of the present invention, to the subject in need thereof. In various embodiments, the method elicits an immune response against YFV.

In various embodiments, the prime dose can be administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously. In various embodiments, the one or more boost dose can be administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously.

In various embodiments, a first of the one or more boost dose can be administered about 2 weeks after one prime dose, or if more than one prime dose then about 2 weeks after the last prime dose.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

FIG. 1 depict various representative versions of the codon-pair deoptimized (CPD) Yellow Fever 17D Viral Genome design.

FIGS. 2A-2C depict a schematic representation of the eight 17D-WWDW genome DNA fragments used in overlapping PCR. 2A. Schematic showing the boundaries of the eight genome fragments. F2 contains the CPD sequences. The green box at the 5′-end of the full-length genome DNA represents the phi-2.5 T7 promoter. 2B. Sizes of eight fragments generated by PCR. 2C. Gel image of the 8 genome fragments amplified from 8 different plasmids.

FIG. 3A depicts a PCR gel check for F1-F8 for construction of the deoptimized YFV. F2 can be either of the wild-type (Wt) or any one of CPD-fragments (DW, WD, DD, or DDDW).

FIG. 3B depicts 17D-WWDW vaccine candidate RT-PCR fragments from Passages 5, 12, and 15 (top, middle, bottom).

FIG. 3C depicts all 8 PCR fragments generated from 17D-WWDW vaccine candidate from viremia sample #18164.

FIG. 4 depicts gel check for four full length CPD YF genome PCR (˜11 kb).

FIG. 5 depicts RNA gel check for four full length YF-CPD genome RNAs.

FIG. 6 plaque assay for the vaccine strain YF-17D (left column) and the recovered YF-DW viral variant (right column) at 33° C. (top row) and 37° C. (bottom row).

FIG. 7 depicts plaque assay for the vaccine strain YF-(left column) and the recovered YF-DDDW viral variant (right column) at 33° C. (top row) and 37° C. (bottom row).

FIGS. 8A-8D depict detection of Infected Vero Cells by Immunohistochemical Staining. Cells transfected with (8A) YF-DD RNA or (8B) no RNA were fixed with Methanol/Acetone 8 days after RNA transfection. Cells infected with (8C) day 4 YF-DD transfection supernatants or (8D) mock supernatant were fixed with Methanol/Acetone 8 days after infection. YF-infected cells were visualized by IHC staining with mouse mAb anti-Flavivirus Group Antigen, clone D1-4G2-4-15 (ATCC® HB-112), in conjunction with HRP-labeled goat anti-mouse secondary antibody and VECTOR VIP chromogenic substrate.

FIG. 9 depicts the gel image of the full-length 17D-WWDW genome DNA constructed by overlapping PCR of 8 genome fragments shown in FIG. 2.

FIG. 10 depicts in vitro transcript of the full-length 17D-WWDW genome RNA generated from overlapping PCR was checked on agarose gel. Ctrl RNA represents RNA used to control for presence of RNase in the gel.

FIG. 11 depicts plaque morphology of the parental 17D and deoptimized 17D-WWDW virus on Vero cells for 5 days at 37° C. Viral titers are 8×10⁵PFU/ml for parental 17D (9th freeze-thaw cycle) and 6.25×10⁷PFU/ml for 17D-WWDW.

FIG. 12 depicts plaque morphology of the parental 17D and deoptimized 17D-WWDW virus on Vero cells for 5 days at 33° C. Viral titers were 8×10⁵PFU/ml for parental 17D (9th freeze-thaw cycle) and 7.75×10⁷PFU/ml for 17D-WWDW.

FIG. 13 depicts neutralizing antibody titers against 17D in monkey sera from Southern Research Study 16128.02. Results are shown as top: mean±SEM, bottom: geomean±geoSD. *p=0.0001, ns; no statistically significant difference, p>0.05. Values below the lowest dilution (<10) were entered as 9 on the graph.

FIG. 14 depicts post-vaccination viremia in NHP serum, as assessed by qRT-PCR.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3^rded., Revised, J. Wiley & Sons (New York, NY 2006); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4^thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N Y 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

A “subject” means any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, pigs, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds. Artificially modified animals include, but are not limited to, SCID mice with human immune systems, outbred or inbred strains of laboratory mice, and athymic nude mice. In a preferred embodiment, the subject is a human. Preferred embodiments of birds are domesticated poultry species, including, but not limited to, chickens, turkeys, ducks, and geese.

A “viral host” means any animal or artificially modified animal, or insect that a virus can infect. Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, pigs, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. In a specific embodiment, the viral host is a human. Embodiments of birds are domesticated poultry species, including, but not limited to, chickens, turkeys, ducks, and geese. Insects include, but are not limited to mosquitos.

“Parent virus” as used herein refer to a reference virus to which a recoded nucleotide sequence is compared for encoding the same or similar amino acid sequence.

“Frequently used codons” or “codon usage bias” as used herein refer to differences in the frequency of occurrence of synonymous codons in coding DNA for a particular species, for example, human, or Yellow Fever Virus.

“Codon pair bias” as used herein refers to synonymous codon pairs that are used more or less frequently than statistically predicted in a particular species, for example, human, or Yellow Fever Virus.

“Corresponding sequence” as used herein refers to a comparison sequence by which the deoptimized sequence is encoding the same or similar amino acid sequence of the comparison sequence. In various embodiments, the corresponding sequence is a sequence that encodes a viral protein. In various embodiments, the corresponding sequence is at least 50 codons in length. In various embodiments, the corresponding sequence is at least 100 codons in length. In various embodiments, the corresponding sequence is at least 150 codons in length. In various embodiments, the corresponding sequence is at least 200 codons in length. In various embodiments, the corresponding sequence is at least 250 codons in length. In various embodiments, the corresponding sequence is at least 300 codons in length. In various embodiments, the corresponding sequence is at least 350 codons in length. In various embodiments, the corresponding sequence is at least 400 codons in length. In various embodiments, the corresponding sequence is at least 450 codons in length. In various embodiments, the corresponding sequence is at least 500 codons in length. In various embodiments, the corresponding sequence is the viral protein sequence. In various embodiments, the corresponding sequence is the sequence of the entire virus.

In various embodiments, “similar amino acid sequence” as used herein refers to an amino acid sequence having less than 2% amino acid substitutions, deletions or additions compared to the comparison sequence. In various embodiments, if specifically provided for in the claims, “similar amino acid sequence” refers to an amino acid sequence having less than 1.75% amino acid substitutions, deletions or additions compared to the comparison sequence. In various embodiments, if specifically provided for in the claims, “similar amino acid sequence” refers to an amino acid sequence having less than 1.5% amino acid substitutions, deletions or additions compared to the comparison sequence. In various embodiments, if specifically provided for in the claims, “similar amino acid sequence” refers to an amino acid sequence having less than 1.25% amino acid substitutions, deletions or additions compared to the comparison sequence. In various embodiments, if specifically provided for in the claims, “similar amino acid sequence” refers to an amino acid sequence having less than 1% amino acid substitutions, deletions or additions compared to the comparison sequence. In various embodiments, if specifically provided for in the claims, “similar amino acid sequence” refers to an amino acid sequence having less than 0.75% amino acid substitutions, deletions or additions compared to the comparison sequence. In various embodiments, if specifically provided for in the claims, “similar amino acid sequence” refers to an amino acid sequence having less than 0.5% amino acid substitutions, deletions or additions compared to the comparison sequence. In various embodiments, if specifically provided for in the claims, “similar amino acid sequence” refers to an amino acid sequence having less than 0.25% amino acid substitutions, deletions or additions compared to the comparison sequence.

Described herein, the inventors used their Synthetic Attenuated Virus Engineering (SAVE) platform to design, generate and test live attenuated vaccine candidates against YF. SAVE is a computational, synthetic biology tool for rapid generation of live-attenuated vaccines. The SAVE computer algorithms provide for the design of virus attenuation by recoding viral genomes rich in underrepresented human codons and codon-pairs without changing the amino acid sequence—a process termed virus deoptimization. The redesigned genome is synthesized de novo and transfected into cells to recover the attenuated, genetically defined, human-cell-deoptimized vaccine strain. The resulting viruses are fully immunogenic, preserving all or essential the epitopes of target virus, but are orders of magnitude attenuated, due to a slowing of viral gene expression in the infected host cell. In various embodiments, resulting viruses are designed or adapted to have particular mutations.

Starting with the published consensus sequence of the current 17D vaccine YF-VAX, we designed and synthesized novel deoptimized vaccine strains, for example, 17D-WWDW and 17D-WD, and each with increasing levels of attenuation over the 17D parental phenotype. One of our candidates, 17D-WWDW, grows to high titers in mammalian cell culture (Vero), is genetically stable for at least 15 passages, and is as immunogenic in Rhesus macaques as the highly effective parental 17D vaccine.

Live attenuated, codon pair deoptimized vaccine candidates against yellow fever, 17D-WWDW and 17D-WD, were successfully recovered following genome reconstruction via overlapping PCR and transfection. 17D-WWDW was genetically stable over 15 passages in Vero cells.

Both deoptimized vaccines were safe and immunogenic in NHPs. Two SC injections of each vaccine tested in the study were well tolerated in the monkeys, and there was no apparent difference in the in-life parameters between the two deoptimized vaccines and the 17D reference vaccine. 17D-WWDW elicited similar levels of neutralizing antibodies to 17D reference vaccine after one dose. Absence of viremia following boost suggests that vaccinated animals were protected from the surrogate challenge posed by the vaccine booster dose. Taken together, these findings suggest that 17D-WWDW is a candidate for an improved, scalable, genetically stable, live attenuated vaccine against YF.

Additionally, we have shown, for example, in International Patent Application No. PCT/US2020/032901, herein incorporated by reference as though fully set forth, that Yellow Fever Virus, particularly, synthetic Yellow Fever Virus Strain 17D can be used for the treatment of cancer. Described herein we develop live attenuated Yellow Fever vaccine candidates that allow manufacture in modern cell culture systems (rather than chicken embryos), while preserving or increasing and stabilizing the attenuation phenotype relative to the current 17D vaccine. To this end codon pair deoptimized cassettes are introduced into the 17D viral genome by reverse genetics methods to “over-attenuate” the resulting vaccine candidate. The over-attenuation provides a safety “buffer” that will allow to absorb potential de-attenuating effects of mutations that may occur upon virus adaptation when switching the manufacturing substrate of the vaccine from chick embryos to cell culture.

Virus Composition, Oncolytic Virus Composition and Pharmaceutical Compositions

Embodiments of the present invention provide for a deoptimized Yellow Fever virus, wherein the E protein coding sequence is deoptimized. Various embodiments of the present invention provide for a pharmaceutical composition comprising a deoptimized Yellow Fever virus wherein the E protein coding sequence is deoptimized and a pharmaceutically acceptable carrier or excipient. In various embodiments, the pharmaceutically acceptable carrier or excipient is sorbitol or gelatin, which can be used as stabilizers. In various embodiments, the composition comprising the deoptimized Yellow Fever virus wherein the E protein coding sequence is deoptimized (e.g., vaccine preparation) can be lyophilized and kept under cold-chain conditions.

In various embodiments, the pharmaceutically acceptable carrier or excipient is particularly adapted for delivery of the deoptimized Yellow Fever virus wherein the E protein coding sequence is deoptimized for cancer treatment; for example, to enhance delivery to the tumor site. Examples of these carriers include but are not limited to carbon nanotube, layered double hydroxide (LDH), iron oxide nanoparticles, mesoporous silica nanoparticles (MSN), polymeric nanoparticles, liposomes, micelle, protein nanoparticles, and dendrimer.

The deoptimized Yellow Fever virus is one which does not cause, or has less than a 0.01% chance of causing Yellow Fever in a mammalian subject and in particular in a human subject.

In various embodiments, the deoptimized Yellow Fever virus wherein the E protein coding sequence is deoptimized is the deoptimized form of Yellow Fever virus (YFV) 17D vaccine (e.g., UniProtKB—P03314 (POLG_YEFV1), as of Jun. 28, 2021, herein incorporated by reference as though fully set forth). In various embodiments, the deoptimized Yellow Fever virus wherein the E protein coding sequence is deoptimized is the deoptimized form of Yellow Fever virus (YFV) 17D (Genbank Accession #JN628279, as of Jun. 28, 2021, Stock et al., 2012; herein incorporated by reference as though fully set forth).

The attenuated live YFV 17D vaccine strain is derived from a wild-type YF virus (the Asibi strain) isolated in Ghana in 1927 and attenuated by serial passages in chicken embryo tissue culture. Two substrains of the 17D vaccine virus are currently used for vaccine production in embryonated chicken eggs, namely 17D-204 and 17DD. Some vaccines are also prepared from a distinct substrain of 17D-204 (17D-213). Thus, in various embodiments, the deoptimized YFV 17D wherein the E protein coding sequence is deoptimized, is deoptimized YFV 17D-204 wherein the E protein coding sequence is deoptimized, deoptimized YFV 17DD wherein the E protein coding sequence is deoptimized, or deoptimized YFV 17D-213 wherein the E protein coding sequence is deoptimized.

Various embodiments of the invention provide a deoptimized YFV virus, which comprises a deoptimized viral genome containing nucleotide substitutions engineered in one or multiple locations in the genome, wherein the substitutions introduce a plurality of synonymous codons into the genome (e.g., codon deoptimization) and/or a change of the order of existing codons for the same amino acid (change of codon pair utilization (e.g., codon-pair deoptimization)). In both cases, the original vaccine strain amino acid sequences are retained. In alternative embodiments, the original vaccine strain amino acid sequences are substantially retained; that is, there is one or more amino acid addition, deletion or substitution in comparison to the original vaccine strain's amino acid sequence. In various embodiments, one or more amino acid addition, deletion or substitution is up to 50 amino acid additions, deletions or substitutions. In various embodiments, one or more amino acid addition, deletion or substitution is up to 40 amino acid additions, deletions or substitutions. In various embodiments, one or more amino acid addition, deletion or substitution is up to 30 amino acid additions, deletions or substitutions. In various embodiments, one or more amino acid addition, deletion or substitution is up to 25 amino acid additions, deletions or substitutions. In various embodiments, one or more amino acid addition, deletion or substitution is up to 20 amino acid additions, deletions or substitutions. In various embodiments, one or more amino acid addition, deletion or substitution is up to 15 amino acid additions, deletions or substitutions. In various embodiments, one or more amino acid addition, deletion or substitution is up to 10 amino acid additions, deletions or substitutions. In various embodiments, one or more amino acid addition, deletion or substitution is up to 5 amino acid additions, deletions or substitutions.

In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about ¾ the length of the E protein. In various embodiments, a continuous segment of E protein is recoded, wherein the continuous segment is about ½ the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about ⅓ the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about ¼ the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about ⅕ the length of the viral protein.

In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 10-20% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 20-30% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 25-35% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 30-40% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 35-45% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 40-50% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 45-55% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 50-60% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 55-65% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 60-70% of the length of the E protein. In various embodiments, a continuous segment of an E protein is recoded, wherein the continuous segment is about 70-80% of the length of the E protein.

Various embodiments of the invention provide for a codon deoptimized yellow fever virus wherein the E protein coding sequence is deoptimized.

In various embodiments, the codon deoptimized yellow fever virus comprises at least 10 deoptimized codons in an E protein coding sequence, wherein the at least 10 deoptimized codons are each a synonymous codon less frequently used in the yellow fever virus. In various embodiments, the codon deoptimized yellow fever virus comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, or 400 deoptimized codons in the E protein coding sequence, wherein the at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, or 400 deoptimized codons are each a synonymous codon less frequently used in the yellow fever virus. The synonymous codon less frequently used in the yellow fever virus is a codon that encodes the same amino acid, but the codon is an unpreferred or less preferred codon by the yellow fever virus for the amino acid.

TABLE 1

Yellow Fever Virus (17D Strain) Codon Usage

Amino

# of
Amino

# of
Amino

# of
Amino

# of

acid
Codon
Use
acid
Codon
Use
acid
Codon
Use
acid
Codon
Use

Phe
UUU
64
Ser
UCU
44
Tyr
UAU
35
Cys
UGU
32

UAC
52

UCC
42

UAC
44

UGC
32

Leu
UUA
8

UCA
56
Ochre
UAA
1
Opal
UGA
0

UUG
72

UCG
8
Amber
UAG
0
Trp
UGG
85

Leu
CUU
46
Pro
CCU
40
His
CAU
50
Arg
CGU
12

CUC
49

CCC
28

CAC
32

CGC
25

CUA
37

CCA
56
Gln
CAA
42

CGA
12

CUG
100

CCG
12

CAG
51

CGG
11

Ile
AUU
63
Thr
ACU
53
Asn
AAU
56
Ser
AGU
34

AUC
69

ACC
51

AAC
68

AGC
31

AUA
44

ACA
73
Lys
AAA
92
Arg
AGA
67

Met
AUG
129

ACG
21

AAG
10

AGG
83

Val
GUU
69
Ala
GCU
83
Asp
GAU
70
Gly
GGU
36

GUC
69

GCC
80

GAC
88

GGC
68

GUA
16

GCA
58
Glu
GAA
108

GGA
124

GUG
132

GCG
23

GAG
102

GGG
73

Codon usage for the yellow fever virus, 17D strain, long open reading frame of 10,233 nucleotides (3411 codons excluding the termination codon). Data from Rice et al. (1985)

In various embodiments, the codon deoptimized yellow fever virus comprises at least 10 deoptimized codons in an E protein coding sequence, wherein the at least 10 deoptimized codons are each a synonymous codon less frequently used in the viral host, such as in humans. In various embodiments, the codon deoptimized yellow fever virus comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, or 400 deoptimized codons in the E protein coding sequence, wherein the at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300 or 400 deoptimized codons are each a synonymous codon less frequently used in the viral host, such as humans. The synonymous codon less frequently used in in the viral host is a codon that encodes the same amino acid, but the codon is an unpreferred codon or less preferred by that viral host for the amino acid. The synonymous codon less frequently used in humans is a codon that encodes the same amino acid, but the codon is an unpreferred codon or less preferred by humans for the amino acid.

In various embodiments, the codon deoptimized yellow fever virus has its E protein coding codons deoptimized and has the same amino acid sequence as YFV 17D, YFV 17D-204, YFV 17DD, or YFV 17D-213. In various embodiments, the codon deoptimized yellow fever virus has its E protein coding codons deoptimized and has up to 1, 2, 3, 4 or 5 amino acid changes as compared to the amino acid sequence as YFV 17D, YFV 17D-204, YFV 17DD, or YFV 17D-213. An amino acid change can be a different amino acid, a deletion of an amino acid, or an addition of an amino acid.

Methods of codon deoptimization are described in International Application No. PCT/US2005/036241, the contents of which are herein incorporated by reference.

Various embodiments of the invention provide for a codon-pair deoptimized (CPD) yellow fever virus wherein the E protein coding sequence is codon-pair deoptimized.

In various embodiments, the codon-pair deoptimized yellow fever virus wherein the E protein coding sequence is codon-pair deoptimized comprises a reduction in codon-pair bias (CPB) as compared to the yellow fever virus before codon-pair deoptimization of the yellow fever virus. Thus, the codon-pair deoptimized yellow fever virus comprises rearranging existing codons in the E protein coding sequence. Rearranging existing codons in the E protein coding sequence comprises substituting a codon pair with a codon pair that has a lower codon-pair score.

As such, it comprises recoded E protein coding sequences wherein each sequence has existing synonymous codons from its parent E protein-coding sequence in a rearranged order and has a CPB less than the CPB of the parent E protein-coding sequence from which it is derived.

In some embodiments, a subset of codon pairs is substituted by rearranging a subset of synonymous codons. In other embodiments, codon pairs are substituted by maximizing the number of rearranged synonymous codons. It is noted that while rearrangement of codons leads to codon-pair bias that is reduced (made more negative) for the virus coding sequence overall, and the rearrangement results in a decreased codon pair scores (CPS) at many locations, there may accompanying CPS increases at other locations, but on average, the codon pair scores, and thus the CPB of the deoptimized sequence, is reduced.

In various embodiments, the CPB is reduced by at least 0.01, at least 0.02, at least 0.03, at least 0.04, at least 0.05, at least 0.10, at least 0.15, at least 0.20, at least 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45 or at least 0.50 compared to the corresponding sequence. In certain embodiments, it is in comparison corresponding sequence from which the calculation is to be made; for example, the corresponding sequence of a wild type virus; or in another example, the corresponding sequence of 17D YFV.

In certain embodiments, the codon pair bias of the recoded sequence is reduced by at least 0.05, or at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, compared to the corresponding sequence. In certain embodiments, it is in comparison corresponding sequence from which the calculation is to be made; for example, the corresponding sequence of a wild type virus; or in another example, the corresponding sequence of 17D YFV.

In embodiments wherein the deoptimized sequence comprises a recoded sequence having reduced codon pair bias compared to a corresponding sequence, the recoded sequence has a codon pair bias less than −0.05, or less than −0.06, or less than −0.07, or less than −0.08, or less than −0.09, or less than −0.1, or less than −0.11, or less than −0.12, or less than −0.13, or less than −0.14, or less than −0.15, or less than −0.16, or less than −0.17, or less than −0.18, or less than −0.19, or less than −0.2, or less than −0.25, or less than −0.3, or less than −0.35, or less than −0.4, or less than −0.45, or less than −0.5.

In various embodiments, the codon pair bias is based on codon pair usage in yellow fever virus. In various embodiments, the codon pair bias is based on codon pair usage in humans.

In various embodiments, the codon-pair deoptimized yellow fever virus wherein the E protein coding sequence is codon-pair deoptimized has the same amino acid sequence as YFV 17D, YFV 17D-204, YFV 17DD, or YFV 17D-213. In various embodiments, the codon-pair deoptimized yellow fever wherein the E protein coding sequence is codon-pair deoptimized virus has up to 1, 2, 3, 4, or 5 amino acid changes as compared to the amino acid sequence as YFV 17D, YFV 17D-204, YFV 17DD, or YFV 17D-213. An amino acid change can be a different amino acid, a deletion of an amino acid, or an addition of an amino acid.

Method of codon-pair deoptimization are described in International Patent Application No. PCT/US2008/058952, the contents of which are herein incorporated by reference.

Various embodiments of the invention provide for a deoptimized yellow fever virus, wherein the frequency of the CG and/or TA (or UA) dinucleotide content in the E protein coding sequence is altered. In various embodiments, the CpG dinucleotide content in the E protein coding sequence in the deoptimized YFV is increased. In various embodiments, the UpA dinucleotide content in the E protein coding sequence in the deoptimized YFV is increased.

For example, the increase is of about 15-55 CpG or UpA di-nucleotides compared the corresponding sequence. In various embodiments, increase is of about 15, 20, 25, 30, 35, 40, 45, or 55 CpG or UpA di-nucleotides compared the corresponding sequence. In some embodiments, the increased number of CpG or UpA di-nucleotides compared to a corresponding sequence is about 10-75, 15-25, 25-50, or 50-75 CpG or UpA di-nucleotides compared the corresponding sequence.

In various embodiments, the deoptimized yellow fever virus wherein the frequency of the CG and/or TA (or UA) dinucleotide content in the E protein coding sequence is altered has the same amino acid sequence as YFV 17D, YFV 17D-204, YFV 17DD, or YFV 17D-213. In various embodiments, the deoptimized yellow fever virus wherein the frequency of the CG and/or TA (or UA) dinucleotide content in the E protein coding sequence is altered has up to 1, 2, 3, 4, or 5 amino acid changes as compared to the amino acid sequence as YFV 17D, YFV 17D-204, YFV 17DD, or YFV 17D-213. An amino acid change can be a different amino acid, a deletion of an amino acid, or an addition of an amino acid.

Methods of altering CG and/or TA (or UA) dinucleotide content are described in International Patent Application No. PCT/US2008/058952, the contents of which are herein incorporated by reference.

In various embodiments, the E protein of the deoptimized YFV is encoded by a polynucleotide having SEQ ID NO:3.

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:3, wherein the variant is not the YFV 17D sequence or wild type sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:3 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3. In various embodiments, the variant of a polynucleotide having SEQ ID NO:3 has up to 20 mutations in SEQ ID NO:3. In various embodiments, the variant of a polynucleotide having SEQ ID NO:3 has up to 10 mutations in SEQ ID NO:3. In various embodiments, the variant of a polynucleotide having SEQ ID NO:3 has up to 5 mutations in SEQ ID NO:3.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:3 encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:3 encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:3 encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:3 encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:3. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence of a polynucleotide having SEQ ID NO:3.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence variant of a polynucleotide having SEQ ID NO:3, wherein the variant is not the YFV 17D sequence. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence variant of a polynucleotide having SEQ ID NO:3, wherein the variant is not the YFV 17D sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:3 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3. In various embodiments, the variant of a polynucleotide having SEQ ID NO:3 has up to 20 mutations in SEQ ID NO:3. In various embodiments, the variant of a polynucleotide having SEQ ID NO:3 has up to 10 mutations in SEQ ID NO:3. In various embodiments, the variant of a polynucleotide having SEQ ID NO:3 has up to 5 mutations in SEQ ID NO:3.

In various embodiments, the E protein of the deoptimized YFV is encoded by a polynucleotide having SEQ ID NO:4.

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:4, wherein the variant is not the YFV 17D sequence or wild type sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:4 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4 In various embodiments, the variant of a polynucleotide having SEQ ID NO:4 has up to 20 mutations in SEQ ID NO:4. In various embodiments, the variant of a polynucleotide having SEQ ID NO:4 has up to 10 mutations in SEQ ID NO:4. In various embodiments, the variant of a polynucleotide having SEQ ID NO:4 has up to 5 mutations in SEQ ID NO:4.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:4 encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:4 encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:4 encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:4 encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:4. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:4.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:4 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4. In various embodiments, the variant of a polynucleotide having SEQ ID NO:4 has up to 20 mutations in SEQ ID NO:4. In various embodiments, the variant of a polynucleotide having SEQ ID NO:4 has up to 10 mutations in SEQ ID NO:4. In various embodiments, the variant of a polynucleotide having SEQ ID NO:4 has up to 5 mutations in SEQ ID NO:4.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO:5.

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:5, wherein the variant is not the YFV 17D sequence or wild type sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:5 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5. In various embodiments, the variant of a polynucleotide having SEQ ID NO:5 has up to 20 mutations in SEQ ID NO:5. In various embodiments, the variant of a polynucleotide having SEQ ID NO:5 has up to 10 mutations in SEQ ID NO:5. In various embodiments, the variant of a polynucleotide having SEQ ID NO:5 has up to 5 mutations in SEQ ID NO:5.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:5 encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:5 encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:5 encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:5 encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:5. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:5.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO:6.

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:6, wherein the variant is not the YFV 17D sequence or wild type sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:6 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:6 In various embodiments, the variant of a polynucleotide having SEQ ID NO:6 has up to 20 mutations in SEQ ID NO:6. In various embodiments, the variant of a polynucleotide having SEQ ID NO:6 has up to 10 mutations in SEQ ID NO:6. In various embodiments, the variant of a polynucleotide having SEQ ID NO:6 has up to 5 mutations in SEQ ID NO:6.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:6 encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:6 encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:6 encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:6 encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:6. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:6.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:6 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:6. In various embodiments, the variant of a polynucleotide having SEQ ID NO:6 has up to 20 mutations in SEQ ID NO:6. In various embodiments, the variant of a polynucleotide having SEQ ID NO:6 has up to 10 mutations in SEQ ID NO:6. In various embodiments, the variant of a polynucleotide having SEQ ID NO:6 has up to 5 mutations in SEQ ID NO:6.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO:7 (YF-WWDW).

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW), wherein the variant is not the YFV 17D sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. In various embodiments, the variant of a polynucleotide having SEQ ID NO:7 has up to 20 mutations in SEQ ID NO:7. In various embodiments, the variant of a polynucleotide having SEQ ID NO:7 has up to 10 mutations in SEQ ID NO:7. In various embodiments, the variant of a polynucleotide having SEQ ID NO:7 has up to 5 mutations in SEQ ID NO:7.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW) encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW) encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW) encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW) encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:7 (YF-WWDW). In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence SEQ ID NO:7 (YF-WWDW).

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with an E protein coding sequence variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW), wherein the variant is not the YFV 17D sequence. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with an E protein coding sequence a variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW), wherein the variant is not the YFV 17D sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:7 (YF-WWDW) has at least 950%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. In various embodiments, the variant of a polynucleotide having SEQ ID NO:7 has up to 20 mutations in SEQ ID NO:7. In various embodiments, the variant of a polynucleotide having SEQ ID NO:7 has up to 10 mutations in SEQ ID NO:7. In various embodiments, the variant of a polynucleotide having SEQ ID NO:7 has up to 5 mutations in SEQ ID NO:7.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO:8.

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:8, wherein the variant is not the YFV 17D sequence or wild type sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:8 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:8 In various embodiments, the variant of a polynucleotide having SEQ ID NO:8 has up to 20 mutations in SEQ ID NO:8. In various embodiments, the variant of a polynucleotide having SEQ ID NO:8 has up to 10 mutations in SEQ ID NO:8. In various embodiments, the variant of a polynucleotide having SEQ ID NO:8 has up to 5 mutations in SEQ ID NO:8.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:8 encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:8 encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:8 encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:8 encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:8. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:8.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:8 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:8. In various embodiments, the variant of a polynucleotide having SEQ ID NO:8 has up to 20 mutations in SEQ ID NO:8. In various embodiments, the variant of a polynucleotide having SEQ ID NO:8 has up to 10 mutations in SEQ ID NO:8. In various embodiments, the variant of a polynucleotide having SEQ ID NO:8 has up to 5 mutations in SEQ ID NO:8.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO:9.

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:9, wherein the variant is not the YFV 17D sequence or wild type sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:9 has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9. In various embodiments, the variant of a polynucleotide having SEQ ID NO:9 has up to 20 mutations in SEQ ID NO:9. In various embodiments, the variant of a polynucleotide having SEQ ID NO:9 has up to 10 mutations in SEQ ID NO:9. In various embodiments, the variant of a polynucleotide having SEQ ID NO:9 has up to 5 mutations in SEQ ID NO:9.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:9 encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:9 encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:9 encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:9 encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:9. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein coding sequence of SEQ ID NO:9.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO: 12 (YF-DW).

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:12 (YF-DW), wherein the variant is not the YFV 17D sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:12 (YF-DW) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 12 has up to 20 mutations in SEQ ID NO:12. In various embodiments, the variant of a polynucleotide having SEQ ID NO:12 has up to 10 mutations in SEQ ID NO:12. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 12 has up to 5 mutations in SEQ ID NO:12.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO: 12 (YF-DW) encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:12 (YF-DW) encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:12 (YF-DW) encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 12 (YF-DW) encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:12 (YF-DW). In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:12 (YF-DW).

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO:13 (YF-WD).

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:13 (YF-DW), wherein the variant is not the YFV 17D sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO:13 (YF-DW) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 13 has up to 20 mutations in SEQ ID NO:13. In various embodiments, the variant of a polynucleotide having SEQ ID NO:13 has up to 10 mutations in SEQ ID NO:13. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 13 has up to 5 mutations in SEQ ID NO:13.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:13 (YF-WD) encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:13 (YF-WD) encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:13 (YF-WD) encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 13 (YF-WD) encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:13 (YF-WD). In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:13 (YF-WD).

In particular embodiments, variant of a polynucleotide having SEQ ID NO:13 (YF-WD) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 13 has up to 20 mutations in SEQ ID NO:13. In various embodiments, the variant of a polynucleotide having SEQ ID NO:13 has up to 10 mutations in SEQ ID NO:13. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 13 has up to 5 mutations in SEQ ID NO:13.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:13 (YF-WD) encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:13 (YF-WD) encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:13 (YF-WD) encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant SEQ ID NO: 13 (YF-WD) encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO: 14 (YF-DD).

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:14 (YF-DD), wherein the variant is not the YFV 17D sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO: 14 (YF-DD) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 14 has up to 20 mutations in SEQ ID NO: 14. In various embodiments, the variant of a polynucleotide having SEQ ID NO:14 has up to 10 mutations in SEQ ID NO:14. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 14 has up to 5 mutations in SEQ ID NO:14.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:14 (YF-DD) encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:14 (YF-DD) encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 14 (YF-DD) encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:14 (YF-DD) encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:14 (YF-DD). In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:14 (YF-DD).

In particular embodiments, variant of a polynucleotide having SEQ ID NO: 14 (YF-DD) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 14 has up to 20 mutations in SEQ ID NO:14. In various embodiments, the variant of a polynucleotide having SEQ ID NO:14 has up to 10 mutations in SEQ ID NO:14. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 14 has up to 5 mutations in SEQ ID NO:14.

In various embodiments, the E protein of the deoptimized YFV is encoded by SEQ ID NO:15 (YF-DDDW).

In various embodiments, the E protein of the deoptimized YFV is encoded by a variant of a polynucleotide having SEQ ID NO:15 (YF-DDDW), wherein the variant is not the YFV 17D sequence.

In particular embodiments, variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:15. In various embodiments, the variant of a polynucleotide having SEQ ID NO:15 has up to 20 mutations in SEQ ID NO:15. In various embodiments, the variant of a polynucleotide having SEQ ID NO:15 has up to 10 mutations in SEQ ID NO: 15. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 15 has up to 5 mutations in SEQ ID NO: 15.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO:15 (YF-DDDW) encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:15 (YF-DDDW) encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO:15 (YF-DDDW) encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW) encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:15 (YF-DDDW). In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein encoded by SEQ ID NO:15 (YF-DDDW).

In various embodiments, the deoptimized YFV is encoded by YFV 17D, wherein the YFV 17D E protein coding sequence is replaced with the E protein encoded by a variant of a polynucleotide having SEQ ID NO:15 (YF-DDDW), wherein the variant is not the YFV 17D sequence. In various embodiments, the deoptimized YFV is encoded by YFV 17D-204, YFV 17DD, or YFV 17D-213, wherein the YFV 17D-204, YFV 17DD, or YFV 17D-213 E protein coding sequence is replaced with the E protein encoded by a variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW), wherein the variant is not the YFV 17D sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW) has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 15 has up to 20 mutations in SEQ ID NO: 15. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 15 has up to 10 mutations in SEQ ID NO: 15. In various embodiments, the variant of a polynucleotide having SEQ ID NO: 15 has up to 5 mutations in SEQ ID NO:15.

In particular embodiments, the variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW) encodes a polypeptide sequence with up to 20, 15, 10, or 5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW) encodes a polypeptide sequence with 10-20 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW) encodes a polypeptide sequence with up to 1-9 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence. In particular embodiments, variant of a polynucleotide having SEQ ID NO: 15 (YF-DDDW) encodes a polypeptide sequence with up to 1-5 amino acid substitutions, deletions or additions as compared to the YFV 17D E protein amino acid sequence.

Wild-type and Deoptimized Yellow Fever Full Length and

Envelope (E) Protein Coding Sequences

SEQ

ID

Sequence
NO:

YF full
AGTAAATCCTGTGTGCTAATTGAGGTGCATTGGTCTGCAAATCGAGTTGCTAGGCA
1

length wt
ATAAACACATTTGGATTAATTTTAATCGTTCGTTGAGCGATTAGCAGAGAACTGAC

genome,
CAGAACATGTCTGGTCGTAAAGCTCAGGGAAAAACCCTGGGCGTCAATATGGTACG

bold is in-
ACGAGGAGTTCGCTCCTTGTCAAACAAAATAAAACAAAAAACAAAACAAATTGGAA

frame E
ACAGACCTGGACCTTCAAGAGGTGTTCAAGGATTTATCTTTTTCTTTTTGTTCAAC

gene
ATTTTGACTGGAAAAAAGATCACAGCCCACCTAAAGAGGTTGTGGAAAATGCTGGA

CCCAAGACAAGGCTTGGCTGTTCTAAGGAAAGTCAAGAGAGTGGTGGCCAGTTTGA

TGAGAGGATTGTCCTCAAGGAAACGCCGTTCCCATGATGTTCTGACTGTGCAATTC

CTAATTTTGGGAATGCTGTTGATGACGGGTGGAGTGACCTTGGTGCGGAAAAACAG

ATGGTTGCTCCTAAATGTGACATCTGAGGACCTCGGGAAAACATTCTCTGTGGGCA

CAGGCAACTGCACAACAAACATTTTGGAAGCCAAGTACTGGTGCCCAGACTCAATG

GAATACAACTGTCCCAATCTCAGTCCAAGAGAGGAGCCAGATGACATTGATTGCTG

GTGCTATGGGGTGGAAAACGTTAGAGTCGCATATGGTAAGTGTGACTCAGCAGGCA

GGTCTAGGAGGTCAAGAAGGGCCATTGACTTGCCTACGCATGAAAACCATGGTTTG

AAGACCCGGCAAGAAAAATGGATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAA

GATTGAGAGATGGTTCGTGAGGAACCCCTTTTTTGCAGTGACGGCTCTGACCATTG

CCTACCTTGTGGGAAGCAACATGACGCAACGAGTCGTGATTGCCCTACTGGTCTTG

GCTGTTGGTCCGGCCTACTCAGCTCACTGCATTGGAATTACTGACAGGGATTTCAT

TGAGGGGGTGCATGGAGGAACTTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTG

TCACTGTTATGGCCCCTGACAAGCCTTCATTGGACATCTCACTAGAGACAGTAGCC

ATTGATAGACCTGCTGAGGTGAGGAAAGTGTGTTACAATGCAGTTCTCACTCATGT

GAAGATTAATGACAAGTGCCCCAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACG

AAGGGGACAATGCGTGCAAGCGCACTTATTCTGATAGAGGCTGGGGCAATGGCTGT

GGCCTATTTGGGAAAGGGAGCATTGTGGCATGCGCCAAATTCACTTGTGCCAAATC

CATGAGTTTGTTTGAGGTTGATCAGACCAAAATTCAGTATGTCATCAGAGCACAAT

TGCATGTAGGGGCCAAGCAGGAAAATTGGACTACCGACATTAAGACTCTCAAGTTT

GATGCCCTGTCAGGCTCCCAGGAAGTCGAGTTCATTGGGTATGGAAAAGCTACACT

GGAATGCCAGGTGCAAACTGCGGTGGACTTTGGTAACAGTTACATCGCTGAGATGG

AAACAGAGAGCTGGATAGTGGACAGACAGTGGGCCCAGGACTTGACCCTGCCATGG

CAGAGTGGAAGTGGCGGGGTGTGGAGAGAGATGCATCATCTTGTCGAATTTGAACC

TCCGCATGCCGCCACTATCAGAGTACTGGCCCTGGGAAACCAGGAAGGCTCCTTGA

AAACAGCTCTTACTGGCGCAATGAGGGTTACAAAGGACACAAATGACAACAACCTT

TACAAACTACATGGTGGACATGTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACT

CAAGGGGACATCCTACAAAATATGCACTGACAAAATGTTTTTTGTCAAGAACCCAA

CTGACACTGGCCATGGCACTGTTGTGATGCAGGTGAAAGTGTCAAAAGGAGCCCCC

TGCAGGATTCCAGTGATAGTAGCTGATGATCTTACAGCGGCAATCAATAAAGGCAT

TTTGGTTACAGTTAACCCCATCGCCTCAACCAATGATGATGAAGTGCTGATTGAGG

TGAACCCACCTTTTGGAGACAGCTACATTATCGTTGGGAGAGGAGATTCACGTCTC

ACTTACCAGTGGCACAAAGAGGGAAGCTCAATAGGAAAGTTGTTCACTCAGACCAT

GAAAGGCGTGGAACGCCTGGCCGTCATGGGAGACACCGCCTGGGATTTCAGCTCCG

CTGGAGGGTTCTTCACTTCGGTTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCC

TTTCAGGGGCTATTTGGCGGCTTGAACTGGATAACAAAGGTCATCATGGGGGCGGT

ACTTATATGGGTTGGCATCAACACAAGAAACATGACAATGTCCATGAGCATGATCT

TGGTAGGAGTGATCATGATGTTTTTGTCTCTAGGAGTTGGGGCGGATCAAGGATGC

GCCATCAACTTTGGCAAGAGAGAGCTCAAGTGCGGAGATGGTATCTTCATATTTAG

AGACTCTGATGACTGGCTGAACAAGTACTCATACTATCCAGAAGATCCTGTGAAGC

TTGCATCAATAGTGAAAGCCTCTTTTGAAGAAGGGAAGTGTGGCCTAAATTCAGTT

GACTCCCTTGAGCATGAGATGTGGAGAAGCAGGGCAGATGAGATCAATGCCATTTT

TGAGGAAAACGAGGTGGACATTTCTGTTGTCGTGCAGGATCCAAAGAATGTTTACC

AGAGAGGAACTCATCCATTTTCCAGAATTCGGGATGGTCTGCAGTATGGTTGGAAG

ACTTGGGGTAAGAACCTTGTGTTCTCCCCAGGGAGGAAGAATGGAAGCTTCATCAT

AGATGGAAAGTCCAGGAAAGAATGCCCGTTTTCAAACCGGGTCTGGAATTCTTTCC

AGATAGAGGAGTTTGGGACGGGAGTGTTCACCACACGCGTGTACATGGACGCAGTC

TTTGAATACACCATAGACTGCGATGGATCTATCTTGGGTGCAGCGGTGAACGGAAA

AAAGAGTGCCCATGGCTCTCCAACATTTTGGATGGGAAGTCATGAAGTAAATGGGA

CATGGATGATCCACACCTTGGAGGCATTAGATTACAAGGAGTGTGAGTGGCCACTG

ACACATACGATTGGAACATCAGTTGAAGAGAGTGAAATGTTCATGCCGAGATCAAT

CGGAGGCCCAGTTAGCTCTCACAATCATATCCCTGGATACAAGGTTCAGACGAACG

GACCTTGGATGCAGGTACCACTAGAAGTGAAGAGAGAAGCTTGCCCAGGGACTAGC

GTGATCATTGATGGCAACTGTGATGGACGGGGAAAATCAACCAGATCCACCACGGA

TAGCGGGAAAGTTATTCCTGAATGGTGTTGCCGCTCCTGCACAATGCCGCCTGTGA

GCTTCCATGGTAGTGATGGGTGTTGGTATCCCATGGAAATTAGGCCAAGGAAAACG

CATGAAAGCCATCTGGTGCGCTCCTGGGTTACAGCTGGAGAAATACATGCTGTCCC

TTTTGGTTTGGTGAGCATGATGATAGCAATGGAAGTGGTCCTAAGGAAAAGACAGG

GACCAAAGCAAATGTTGGTTGGAGGAGTAGTGCTCTTGGGAGCAATGCTGGTCGGG

CAAGTAACTCTCCTTGATTTGCTGAAACTCACAGTGGCTGTGGGATTGCATTTCCA

TGAGATGAACAATGGAGGAGACGCCATGTATATGGCGTTGATTGCTGCCTTTTCAA

TCAGACCAGGGCTGCTCATCGGCTTTGGGCTCAGGACCCTATGGAGCCCTCGGGAA

CGCCTTGTGCTGACCCTAGGAGCAGCCATGGTGGAGATTGCCTTGGGTGGCGTGAT

GGGCGGCCTGTGGAAGTATCTAAATGCAGTTTCTCTCTGCATCCTGACAATAAATG

CTGTTGCTTCTAGGAAAGCATCAAATACCATCTTGCCCCTCATGGCTCTGTTGACA

CCTGTCACTATGGCTGAGGTGAGACTTGCCGCAATGTTCTTTTGTGCCGTGGTTAT

CATAGGGGTCCTTCACCAGAATTTCAAGGACACCTCCATGCAGAAGACTATACCTC

TGGTGGCCCTCACACTCACATCTTACCTGGGCTTGACACAACCTTTTTTGGGCCTG

TGTGCATTTCTGGCAACCCGCATATTTGGGCGAAGGAGTATCCCAGTGAATGAGGC

ACTCGCAGCAGCTGGTCTAGTGGGAGTGCTGGCAGGACTGGCTTTTCAGGAGATGG

AGAACTTCCTTGGTCCGATTGCAGTTGGAGGACTCCTGATGATGCTGGTTAGCGTG

GCTGGGAGGGTGGATGGGCTAGAGCTCAAGAAGCTTGGTGAAGTTTCATGGGAAGA

GGAGGCGGAGATCAGCGGGAGTTCCGCCCGCTATGATGTGGCACTCAGTGAACAAG

GGGAGTTCAAGCTGCTTTCTGAAGAGAAAGTGCCATGGGACCAGGTTGTGATGACC

TCGCTGGCCTTGGTTGGGGCTGCCCTCCATCCATTTGCTCTTCTGCTGGTCCTTGC

TGGGTGGCTGTTTCATGTCAGGGGAGCTAGGAGAAGTGGGGATGTCTTGTGGGATA

TTCCCACTCCTAAGATCATCGAGGAATGTGAACATCTGGAGGATGGGATTTATGGC

ATATTCCAGTCAACCTTCTTGGGGGCCTCCCAGCGAGGAGTGGGAGTGGCACAGGG

AGGGGTGTTCCACACAATGTGGCATGTCACAAGAGGAGCTTTCCTTGTCAGGAATG

GCAAGAAGTTGATTCCATCTTGGGCTTCAGTAAAGGAAGACCTTGTCGCCTATGGT

GGCTCATGGAAGTTGGAAGGCAGATGGGATGGAGAGGAAGAGGTCCAGTTGATCGC

GGCTGTTCCAGGAAAGAACGTGGTCAACGTCCAGACAAAACCGAGCTTGTTCAAAG

TGAGGAATGGGGGAGAAATCGGGGCTGTCGCTCTTGACTATCCGAGTGGCACTTCA

GGATCTCCTATTGTTAACAGGAACGGAGAGGTGATTGGGCTGTACGGCAATGGCAT

CCTTGTCGGTGACAACTCCTTCGTGTCCGCCATATCCCAGACTGAGGTGAAGGAAG

AAGGAAAGGAGGAGCTCCAAGAGATCCCGACAATGCTAAAGAAAGGAATGACAACT

GTCCTTGATTTTCATCCTGGAGCTGGGAAGACAAGACGTTTCCTCCCACAGATCTT

GGCCGAGTGCGCACGGAGACGCTTGCGCACTCTTGTGTTGGCCCCCACCAGGGTTG

TTCTTTCTGAAATGAAGGAGGCTTTTCACGGCCTGGACGTGAAATTCCACACACAG

GCTTTTTCCGCTCACGGCAGCGGGAGAGAAGTCATTGATGCTATGTGCCATGCCAC

CCTAACTTACAGGATGTTGGAACCAACTAGGGTTGTTAACTGGGAAGTGATCATTA

TGGATGAAGCCCATTTTTTGGATCCAGCTAGCATAGCCGCTAGAGGTTGGGCAGCG

CACAGAGCTAGGGCAAATGAAAGTGCAACAATCTTGATGACAGCCACACCGCCTGG

GACTAGTGATGAATTTCCACATTCAAATGGTGAAATAGAAGATGTTCAAACGGACA

TACCCAGTGAGCCCTGGAACACAGGGCATGACTGGATCCTGGCTGACAAAAGGCCC

ACGGCATGGTTCCTTCCATCCATCAGAGCTGCAAATGTCATGGCTGCCTCTTTGCG

TAAGGCTGGAAAGAGTGTGGTGGTCCTGAACAGGAAAACCTTTGAGAGAGAATACC

CCACGATAAAGCAGAAGAAACCTGACTTTATATTGGCCACTGACATAGCTGAAATG

GGAGCCAACCTTTGCGTGGAGCGAGTGCTGGATTGCAGGACGGCTTTTAAGCCTGT

GCTTGTGGATGAAGGGAGGAAGGTGGCAATAAAAGGGCCACTTCGTATCTCCGCAT

CCTCTGCTGCTCAAAGGAGGGGGCGCATTGGGAGAAATCCCAACAGAGATGGAGAC

TCATACTACTATTCTGAGCCTACAAGTGAAAATAATGCCCACCACGTCTGCTGGTT

GGAGGCCTCAATGCTCTTGGACAACATGGAGGTGAGGGGTGGAATGGTCGCCCCAC

TCTATGGCGTTGAAGGAACTAAAACACCAGTTTCCCCTGGTGAAATGAGACTGAGG

GATGACCAGAGGAAAGTCTTCAGAGAACTAGTGAGGAATTGTGACCTGCCCGTTTG

GCTTTCGTGGCAAGTGGCCAAGGCTGGTTTGAAGACGAATGATCGTAAGTGGTGTT

TTGAAGGCCCTGAGGAACATGAGATCTTGAATGACAGCGGTGAAACAGTGAAGTGC

AGGGCTCCTGGAGGAGCAAAGAAGCCTCTGCGCCCAAGGTGGTGTGATGAAAGGGT

GTCATCTGACCAGAGTGCGCTGTCTGAATTTATTAAGTTTGCTGAAGGTAGGAGGG

GAGCTGCTGAAGTGCTAGTTGTGCTGAGTGAACTCCCTGATTTCCTGGCTAAAAAA

GGTGGAGAGGCAATGGATACCATCAGTGTGTTTCTCCACTCTGAGGAAGGCTCTAG

GGCTTACCGCAATGCACTATCAATGATGCCTGAGGCAATGACAATAGTCATGCTGT

TTATACTGGCTGGACTACTGACATCGGGAATGGTCATCTTTTTCATGTCTCCCAAA

GGCATCAGTAGAATGTCTATGGCGATGGGCACAATGGCCGGCTGTGGATATCTCAT

GTTCCTTGGAGGCGTCAAACCCACTCACATCTCCTATATCATGCTCATATTCTTTG

TCCTGATGGTGGTTGTGATCCCCGAGCCAGGGCAACAAAGGTCCATCCAAGACAAC

CAAGTGGCATACCTCATTATTGGCATCCTGACGCTGGTTTCAGCGGTGGCAGCCAA

CGAGCTAGGCATGCTGGAGAAAACCAAAGAGGACCTCTTTGGGAAGAAGAACTTAA

TTCCATCTAGTGCTTCACCCTGGAGTTGGCCGGATCTTGACCTGAAGCCAGGAGCT

GCCTGGACAGTGTACGTTGGCATTGTTACAATGCTCTCTCCAATGTTGCACCACTG

GATCAAAGTCGAATATGGCAACCTGTCTCTGTCTGGAATAGCCCAGTCAGCCTCAG

TCCTTTCTTTCATGGACAAGGGGATACCATTCATGAAGATGAATATCTCGGTCATA

ATGCTGCTGGTCAGTGGCTGGAATTCAATAACAGTGATGCCTCTGCTCTGTGGCAT

AGGGTGCGCCATGCTCCACTGGTCTCTCATTTTACCTGGAATCAAAGCGCAGCAGT

CAAAGCTTGCACAGAGAAGGGTGTTCCATGGCGTTGCCAAGAACCCTGTGGTTGAT

GGGAATCCAACAGTTGACATTGAGGAAGCTCCTGAAATGCCTGCCCTTTATGAGAA

GAAACTGGCTCTATATCTCCTTCTTGCTCTCAGCCTAGCTTCTGTTGCCATGTGCA

GAACGCCCTTTTCATTGGCTGAAGGCATTGTCCTAGCATCAGCTGCCCTAGGGCCG

CTCATAGAGGGAAACACCAGCCTTCTTTGGAATGGACCCATGGCTGTCTCCATGAC

AGGAGTCATGAGGGGGAATCACTATGCTTTTGTGGGAGTCATGTACAATCTATGGA

AGATGAAAACTGGACGCCGGGGGAGCGCGAATGGAAAAACTTTGGGTGAAGTCTGG

AAGAGGGAACTGAATCTGTTGGACAAGCGACAGTTTGAGTTGTATAAAAGGACCGA

CATTGTGGAGGTGGATCGTGATACGGCACGCAGGCATTTGGCCGAAGGGAAGGTGG

ACACCGGGGTGGCGGTCTCCAGGGGGACCGCAAAGTTAAGGTGGTTCCATGAGCGT

GGCTATGTCAAGCTGGAAGGTAGGGTGATTGACCTGGGGTGTGGCCGCGGAGGCTG

GTGTTACTACGCTGCTGCGCAAAAGGAAGTGAGTGGGGTCAAAGGATTTACTCTTG

GAAGAGACGGCCATGAGAAACCCATGAATGTGCAAAGTCTGGGATGGAACATCATC

ACCTTCAAGGACAAAACTGATATCCACCGCCTAGAACCAGTGAAATGTGACACCCT

TTTGTGTGACATTGGAGAGTCATCATCGTCATCGGTCACAGAGGGGGAAAGGACCG

TGAGAGTTCTTGATACTGTAGAAAAATGGCTGGCTTGTGGGGTTGACAACTTCTGT

GTGAAGGTGTTAGCTCCATACATGCCAGATGTTCTCGAGAAACTGGAATTGCTCCA

AAGGAGGTTTGGCGGAACAGTGATCAGGAACCCTCTCTCCAGGAATTCCACTCATG

AAATGTACTACGTGTCTGGAGCCCGCAGCAATGTCACATTTACTGTGAACCAAACA

TCCCGCCTCCTGATGAGGAGAATGAGGCGTCCAACTGGAAAAGTGACCCTGGAGGC

TGACGTCATCCTCCCAATTGGGACACGCAGTGTTGAGACAGACAAGGGACCCCTGG

ACAAAGAGGCCATAGAAGAAAGGGTTGAGAGGATAAAATCTGAGTACATGACCTCT

TGGTTTTATGACAATGACAACCCCTACAGGACCTGGCACTACTGTGGCTCCTATGT

CACAAAAACCTCAGGAAGTGCGGCGAGCATGGTAAATGGTGTTATTAAAATTCTGA

CATATCCATGGGACAGGATAGAGGAGGTCACAAGAATGGCAATGACTGACACAACC

CCTTTTGGACAGCAAAGAGTGTTTAAAGAAAAAGTTGACACCAGAGCAAAGGATCC

ACCAGCGGGAACTAGGAAGATCATGAAAGTTGTCAACAGGTGGCTGTTCCGCCACC

TGGCCAGAGAAAAGAACCCCAGACTGTGCACAAAGGAAGAATTTATTGCAAAAGTC

CGAAGTCATGCAGCCATTGGAGCTTACCTGGAAGAACAAGAACAGTGGAAGACTGC

CAATGAGGCTGTCCAAGACCCAAAGTTCTGGGAACTGGTGGATGAAGAAAGGAAGC

TGCACCAACAAGGCAGGTGTCGGACTTGTGTGTACAACATGATGGGGAAAAGAGAG

AAGAAGCTGTCAGAGTTTGGGAAAGCAAAGGGAAGCCGTGCCATATGGTATATGTG

GCTGGGAGCGCGGTATCTTGAGTTTGAGGCCCTGGGATTCCTGAATGAGGACCATT

GGGCTTCCAGGGAAAACTCAGGAGGAGGAGTGGAAGGCATTGGCTTACAATACCTA

GGATATGTGATCAGAGACCTGGCTGCAATGGATGGTGGTGGATTCTACGCGGATGA

CACCGCTGGATGGGACACGCGCATCACAGAGGCAGACCTTGATGATGAACAGGAGA

TCTTGAACTACATGAGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATG

ACATACAAGAACAAAGTGGTGAAAGTGTTGAGACCAGCCCCAGGAGGGAAAGCCTA

CATGGATGTCATAAGTCGACGAGACCAGAGAGGATCCGGGCAGGTAGTGACTTATG

CTCTGAACACCATCACCAACTTGAAAGTCCAATTGATCAGAATGGCAGAAGCAGAG

ATGGTGATACATCACCAACATGTTCAAGATTGTGATGAATCAGTTCTGACCAGGCT

GGAGGCATGGCTCACTGAGCACGGATGTAACAGACTGAAGAGGATGGCGGTGAGTG

GAGACGACTGTGTGGTCCGGCCCATCGATGACAGGTTCGGCCTGGCCCTGTCCCAT

CTCAACGCCATGTCCAAGGTTAGAAAGGACATATCTGAATGGCAGCCATCAAAAGG

GTGGAATGATTGGGAGAATGTGCCCTTCTGTTCCCACCACTTCCATGAACTACAGC

TGAAGGATGGCAGGAGGATTGTGGTGCCTTGCCGAGAACAGGACGAGCTCATTGGG

AGAGGAAGGGTGTCTCCAGGAAACGGCTGGATGATCAAGGAAACAGCTTGCCTCAG

CAAAGCCTATGCCAACATGTGGTCACTGATGTATTTTCACAAAAGGGACATGAGGC

TACTGTCATTGGCTGTTTCCTCAGCTGTTCCCACCTCATGGGTTCCACAAGGACGC

ACAACATGGTCGATTCATGGGAAAGGGGAGTGGATGACCACGGAAGACATGCTTGA

GGTGTGGAACAGAGTATGGATAACCAACAACCCACACATGCAGGACAAGACAATGG

TGAAAAAATGGAGAGATGTCCCTTATCTAACCAAGAGACAAGACAAGCTGTGCGGA

TCACTGATTGGAATGACCAATAGGGCCACCTGGGCCTCCCACATCCATTTGGTCAT

CCATCGTATCCGAACGCTGATTGGACAGGAGAAATACACTGACTACCTAACAGTCA

TGGACAGGTATTCTGTGGATGCTGACCTGCAACTGGGTGAGCTTATCTGAAACACC

ATCTAACAGGAATAACCGGGATACAAACCACGGGTGGAGAACCGGACTCCCCACAA

CCTGAAACCGGGATATAAACCACGGCTGGAGAACCGGACTCCGCACTTAAAATGAA

ACAGAAACCGGGATAAAAACTACGGATGGAGAACCGGACTCCACACATTGAGACAG

AAGAAGTTGTCAGCCCAGAACCCCACACGAGTTTTGCCACTGCTAAGCTGTGAGGC

AGTGCAGGCTGGGACAGCCGACCTCCAGGTTGCGAAAAACCTGGTTTCTGGGACCT

CCCACCCCAGAGTAAAAAGAACGGAGCCTCCGCTACCACCCTCCCACGTGGTGGTA

GAAAGACGGGGTCTAGAGGTTAGAGGAGACCCTCCAGGGAACAAATAGTGGGACCA

TATTGACGCCAGGGAAAGACCGGAGTGGTTCTCTGCTTTTCCTCCAGAGGTCTGTG

AGCACAGTTTGCTCAAGAATAAGCAGACCTTTGGATGACAAACACAAAACCACT

YF wt E-
GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAAC
2

gene (1479
TTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGACA

nts)
AGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGTG

AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCC

CAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCAAGC

GCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAAGGGAGC

ATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTGAGGTTGA

TCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGCCAAGCAGG

AAAATTGGACTACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAGGCTCCCAG

GAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGC

GGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGG

ACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTG

TGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAG

AGTACTGGCCCTGGGAAACCAGGAAGGCTCCTTGAAAACAGCTCTTACTGGCGCAA

TGAGGGTTACAAAGGACACAAATGACAACAACCTTTACAAACTACATGGTGGACAT

GTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCAAGGGGACATCCTACAAAAT

ATGCACTGACAAAATGTTTTTTGTCAAGAACCCAACTGACACTGGCCATGGCACTG

TTGTGATGCAGGTGAAAGTGTCAAAAGGAGCCCCCTGCAGGATTCCAGTGATAGTA

GCTGATGATCTTACAGCGGCAATCAATAAAGGCATTTTGGTTACAGTTAACCCCAT

CGCCTCAACCAATGATGATGAAGTGCTGATTGAGGTGAACCCACCTTTTGGAGACA

GCTACATTATCGTTGGGAGAGGAGATTCACGTCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCG

YF
GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAAC
3

E-gene
TTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGACA

WD (1479
AGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGTG

nts, lower-
AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCC

case
CAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCAAGC

represents
GCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAAGGGAGC

CPD
ATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTGAGGTTGA

region)
TCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGCCAAGCAGG

AAAATTGGACTACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAGGCTCCCAG

GAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGC

GGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGG

ACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTG

TGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAG

AGTACTGGCCCTGGGAAACCAGGAAGGCTCCcttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagactgacataccaatggcataaagag

ggatcgtcaatcggtaagttgtttacacagactatgaaaggggtggagagattggc

cgttatgggcgataccgcttgggactttagttccgccggagggttttttactagcg

tcggaaaggggatacataccgtattcggatccgcttttcaggggttgttcggcgga

ctgaattggattacgaaagtgattatgggcgccgtacttatttgggtggggattaa

cactaggaatatgactatgtctatgtctatgatactagtcggagtgattatgatgt

ttctgtcattgggcgtaggcgct

YF
GCTCACTGCATTGGAATTACTGACAGGGATtttatcgagggggtgcatggcggaac
4

E-gene
ttgggttagcgctacactcgaacaggacaaatgcgttaccgttatggcccccgata

DW (1479
agcctagcctagacattagtctcgaaaccgttgcgatcgatagacccgccgaagtg

nts, lower-
agaaaagtgtgttataacgccgtactgactcacgttaagattaacgacaaatgccc

case
tagtacaggcgaagcgcatctagccgaagagaacgagggcgataacgcatgcaaac

represents
gtacttatagcgatagggggtgggggaacggatgcggattgttcggtaaggggtca

CPD
atcgtcgcatgcgctaagtttacatgcgctaagtctatgtcattgttcgaagtcga

region)
tcagactaagattcagtacgtgattagagcgcaattgcatgtgggagcgaaacagg

agaattggactactgacattaagacactgaaattcgacgcccttagcggatcacag

gaggtcgagtttattgggtacggaaaagcgacactcgagtgtcaggtgcagactgc

cgttgactttggcaattcatacatagccgaaatggagacagagtcatggatcgttg

acagacagtgggcccaggatctgacattgccatggcaatccggatccggaggcgtt

tggcgcgaaatgcatcatctagtcgagttcgaaccgccacatgccgctacaatcag

agtgttggccctaggcaatcaggagggaTCCTTGAAAACAGCTCTTACTGGCGCAA

TGAGGGTTACAAAGGACACAAATGACAACAACCTTTACAAACTACATGGTGGACAT

GTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCAAGGGGACATCCTACAAAAT

ATGCACTGACAAAATGTTTTTTGTCAAGAACCCAACTGACACTGGCCATGGCACTG

TTGTGATGCAGGTGAAAGTGTCAAAAGGAGCCCCCTGCAGGATTCCAGTGATAGTA

GCTGATGATCTTACAGCGGCAATCAATAAAGGCATTTTGGTTACAGTTAACCCCAT

CGCCTCAACCAATGATGATGAAGTGCTGATTGAGGTGAACCCACCTTTTGGAGACA

GCTACATTATCGTTGGGAGAGGAGATTCACGTCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCG

YF
GCTCACTGCATTGGAATTACTGACAGGGATtttatcgagggggtgcatggcggaac
5

E-gene
ttgggttagcgctacactcgaacaggacaaatgcgttaccgttatggcccccgata

DD (1479
agcctagcctagacattagtctcgaaaccgttgcgatcgatagacccgccgaagtg

nts, lower-
agaaaagtgtgttataacgccgtactgactcacgttaagattaacgacaaatgccc

case
tagtacaggcgaagcgcatctagccgaagagaacgagggcgataacgcatgcaaac

represents
gtacttatagcgatagggggtgggggaacggatgcggattgttcggtaaggggtca

CPD
atcgtcgcatgcgctaagtttacatgcgctaagtctatgtcattgttcgaagtcga

region)
tcagactaagattcagtacgtgattagagcgcaattgcatgtgggagcgaaacagg

agaattggactactgacattaagacactgaaattcgacgcccttagcggatcacag

gaggtcgagtttattgggtacggaaaagcgacactcgagtgtcaggtgcagactgc

cgttgactttggcaattcatacatagccgaaatggagacagagtcatggatcgttg

acagacagtgggcccaggatctgacattgccatggcaatccggatccggaggcgtt

tggcgcgaaatgcatcatctagtcgagttcgaaccgccacatgccgctacaatcag

agtgttggccctaggcaatcaggagggatcccttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagactgacataccaatggcataaagag

ggatcgtcaatcggtaagttgtttacacagactatgaaaggggtggagagattggc

cgttatgggcgataccgcttgggactttagttccgccggagggttttttactagcg

tcggaaaggggatacataccgtattcggatccgcttttcaggggttgttcggcgga

ctgaattggattacgaaagtgattatgggcgccgtacttatttgggtggggattaa

cactaggaatatgactatgtctatgtctatgatactagtcggagtgattatgatgt

ttctgtcattgggcgtaggcgct

YF
GCTCACTGCATTGGAATTACTGACAGGGATtttatcgagggggtgcatggcggaac
6

E-gene
ttgggttagcgctacactcgaacaggacaaatgcgttaccgttatggcccccgata

DDDW
agcctagcctagacattagtctcgaaaccgttgcgatcgatagacccgccgaagtg

(1479 nts,
agaaaagtgtgttataacgccgtactgactcacgttaagattaacgacaaatgccc

lower-case
tagtacaggcgaagcgcatctagccgaagagaacgagggcgataacgcatgcaaac

represents
gtacttatagcgatagggggtgggggaacggatgcggattgttcggtaaggggtca

CPD
atcgtcgcatgcgctaagtttacatgcgctaagtctatgtcattgttcgaagtcga

region)
tcagactaagattcagtacgtgattagagcgcaattgcatgtgggagcgaaacagg

agaattggactactgacattaagacactgaaattcgacgcccttagcggatcacag

gaggtcgagtttattgggtacggaaaagcgacactcgagtgtcaggtgcagactgc

cgttgactttggcaattcatacatagccgaaatggagacagagtcatggatcgttg

acagacagtgggcccaggatctgacattgccatggcaatccggatccggaggcgtt

tggcgcgaaatgcatcatctagtcgagttcgaaccgccacatgccgctacaatcag

agtgttggccctaggcaatcaggagggatcccttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagaCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGGGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCG

YF
GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAAC
7

E-gene
TTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGACA

WWDW
AGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGTG

(1479 nts,
AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCC

lower-case
CAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCAAGC

represents
GCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAAGGGAGC

CPD
ATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTGAGGTTGA

region)
TCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGCCAAGCAGG

AAAATTGGACTACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAGGCTCCCAG

GAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGC

GGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGG

ACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTG

TGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAG

AGTACTGGCCCTGGGAAACCAGGAAGGCTCCcttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagaCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCG

E-gene of
GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAAC
8

YF-17D
TTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGACA

WD-E-
AGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGTG

153N (E-
AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCC

153N
CAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCAAGC

codon is in
GCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAAGGGAGC

bold,
ATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTGAGGTTGA

lower-case
TCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGCCAAGCAGG

represents
AAAATTGGAATACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAGGCTCCCAG

CPD
GAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGC

region)
GGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGG

ACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTG

TGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAG

AGTACTGGCCCTGGGAAACCAGGAAGGCTCCcttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagactgacataccaatggcataaagag

ggatcgtcaatcggtaagttgtttacacagactatgaaaggggtggagagattggc

cgttatgggcgataccgcttgggactttagttccgccggagggttttttactagcg

tcggaaaggggatacataccgtattcggatccgcttttcaggggttgttcggcgga

ctgaattggattacgaaagtgattatgggcgccgtacttatttgggtggggattaa

cactaggaatatgactatgtctatgtctatgatactagtcggagtgattatgatgt

ttctgtcattgggcgtaggcgct

E-gene of
GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAAC
9

YF-17D
TTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGACA

WWDW-
AGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGTG

E-153N (E-
AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCC

153N
CAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCAAGC

codon is in
GCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAAGGGAGC

bold,
ATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTGAGGTTGA

lower-case
TCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGCCAAGCAGG

represents
AAAATTGGAATACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAGGCTCCCAG

CPD
GAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGC

region)
GGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGG

ACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTG

TGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAG

AGTACTGGCCCTGGGAAACCAGGAAGGCTCCcttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagaCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCG

YF Wt
AHCIGITDRDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEV
10

E-protein
RKVCYNAVLTHVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGS

sequences.
IVACAKFTCAKSMSLFEVDQTKIQYVIRAQLHVGAKQENWTTDIKTLKEDALSGSQ

493 AAs,
EVEFIGYGKATLECQVQTAVDEGNSYIAEMETESWIVDRQWAQDLTLPWQSGSGGV

E153=T is
WREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTKDTNDNNLYKLHGGH

bolded.
VSCRVKLSALTLKGTSYKICTDKMFFVKNPTDTGHGTVVMQVKVSKGAPCRIPVIV

E-protein
ADDLTAAINKGILVTVNPIASTNDDEVLIEVNPPFGDSYIIVGRGDSRLTYQWHKE

sequences
GSSIGKLFTQTMKGVERLAVMGDTAWDESSAGGFFTSVGKGIHTVFGSAFQGLEGG

of E-WD,
LNWITKVIMGAVLIWVGINTRNMTMSMSMILVGVIMMELSLGVGA

E-DW, E-

DD, E-

WWDW,

and E-

DDDW are

the same as

this

sequence.

YF-17D
AHCIGITDRDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEV
11

WD-E-
RKVCYNAVLTHVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGS

153N
IVACAKFTCAKSMSLFEVDQTKIQYVIRAQLHVGAKQENWNTDIKTLKEDALSGSQ

and
EVEFIGYGKATLECQVQTAVDEGNSYIAEMETESWIVDRQWAQDLTLPWQSGSGGV

YF-17D
WREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTKDTNDNNLYKLHGGH

WWDW-
VSCRVKLSALTLKGTSYKICTDKMFFVKNPTDTGHGTVVMQVKVSKGAPCRIPVIV

E-153N
ADDLTAAINKGILVTVNPIASTNDDEVLIEVNPPFGDSYIIVGRGDSRLTYQWHKE

E-protein
GSSIGKLFTQTMKGVERLAVMGDTAWDESSAGGFFTSVGKGIHTVFGSAFQGLEGG

sequences.
LNWITKVIMGAVLIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA

493 AAs,

E153=N is

bolded.

YF-Env-
ATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGATGGTTCGTGAG
12

DW
GAACCCCTTTTTTGCAGTGACGGCTCTGACCATTGCCTACCTTGTGGGAAGCAACA

TGACGCAACGAGTCGTGATTGCCCTACTGGTCTTGGCTGTTGGTCCGGCCTACTCA

GCTCACTGCATTGGAATTACTGACAGGGATtttatcgagggggtgcatggcggaac

ttgggttagcgctacactcgaacaggacaaatgcgttaccgttatggcccccgata

agcctagcctagacattagtctcgaaaccgttgcgatcgatagacccgccgaagtg

agaaaagtgtgttataacgccgtactgactcacgttaagattaacgacaaatgccc

tagtacaggcgaagcgcatctagccgaagagaacgagggcgataacgcatgcaaac

gtacttatagcgatagggggtgggggaacggatgcggattgttcggtaaggggtca

atcgtcgcatgcgctaagtttacatgcgctaagtctatgtcattgttcgaagtcga

tcagactaagattcagtacgtgattagagcgcaattgcatgtgggagcgaaacagg

agaattggactactgacattaagacactgaaattcgacgcccttagcggatcacag

gaggtcgagtttattgggtacggaaaagcgacactcgagtgtcaggtgcagactgc

cgttgactttggcaattcatacatagccgaaatggagacagagtcatggatcgttg

acagacagtgggcccaggatctgacattgccatggcaatccggatccggaggcgtt

tggcgcgaaatgcatcatctagtcgagttcgaaccgccacatgccgctacaatcag

agtgttggccctaggcaatcaggagggaTCCTTGAAAACAGCTCTTACTGGCGCAA

TGAGGGTTACAAAGGACACAAATGACAACAACCTTTACAAACTACATGGTGGACAT

GTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCAAGGGGACATCCTACAAAAT

ATGCACTGACAAAATGTTTTTTGTCAAGAACCCAACTGACACTGGCCATGGCACTG

TTGTGATGCAGGTGAAAGTGTCAAAAGGAGCCCCCTGCAGGATTCCAGTGATAGTA

GCTGATGATCTTACAGCGGCAATCAATAAAGGCATTTTGGTTACAGTTAACCCCAT

CGCCTCAACCAATGATGATGAAGTGCTGATTGAGGTGAACCCACCTTTTGGAGACA

GCTACATTATCGTTGGGAGAGGAGATTCACGTCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCGGATCAAGGATGCGCCATCAACTTTGGCAAGAGA

GAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCTGATGACTGGCTGAA

CAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCT

CT

YF-Env-
ATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGATGGTTCGTGAG
13

WD
GAACCCCTTTTTTGCAGTGACGGCTCTGACCATTGCCTACCTTGTGGGAAGCAACA

E gene
TGACGCAACGAGTCGTGATTGCCCTACTGGTCTTGGCTGTTGGTCCGGCCTACTCA

within this
GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAAC

sequence
TTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGACA

AGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGTG

AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCC

CAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCAAGC

GCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAAGGGAGC

ATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTGAGGTTGA

TCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGCCAAGCAGG

AAAATTGGACTACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAGGCTCCCAG

GAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGC

GGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGG

ACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTG

TGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAG

AGTACTGGCCCTGGGAAACCAGGAAGGCTCCcttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagactgacataccaatggcataaagag

ggatcgtcaatcggtaagttgtttacacagactatgaaaggggtggagagattggc

cgttatgggcgataccgcttgggactttagttccgccggagggttttttactagcg

tcggaaaggggatacataccgtattcggatccgcttttcaggggttgttcggcgga

ctgaattggattacgaaagtgattatgggcgccgtacttatttgggtggggattaa

cactaggaatatgactatgtctatgtctatgatactagtcggagtgattatgatgt

ttctgtcattgggcgtaggcgctGATCAAGGATGCGCCATCAACTTTGGCAAGAGA

GAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCTGATGACTGGCTGAA

CAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCT

CT

YF-Env-
ATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGATGGTTCGTGAG
14

DD
GAACCCCTTTTTTGCAGTGACGGCTCTGACCATTGCCTACCTTGTGGGAAGCAACA

E gene
TGACGCAACGAGTCGTGATTGCCCTACTGGTCTTGGCTGTTGGTCCGGCCTACTCA

within this
GCTCACTGCATTGGAATTACTGACAGGGATtttatcgagggggtgcatggcggaac

sequence
ttgggttagcgctacactcgaacaggacaaatgcgttaccgttatggcccccgata

agcctagcctagacattagtctcgaaaccgttgcgatcgatagacccgccgaagtg

agaaaagtgtgttataacgccgtactgactcacgttaagattaacgacaaatgccc

tagtacaggcgaagcgcatctagccgaagagaacgagggcgataacgcatgcaaac

gtacttatagcgatagggggtgggggaacggatgcggattgttcggtaaggggtca

atcgtcgcatgcgctaagtttacatgcgctaagtctatgtcattgttcgaagtcga

tcagactaagattcagtacgtgattagagcgcaattgcatgtgggagcgaaacagg

agaattggactactgacattaagacactgaaattcgacgcccttagcggatcacag

gaggtcgagtttattgggtacggaaaagcgacactcgagtgtcaggtgcagactgc

cgttgactttggcaattcatacatagccgaaatggagacagagtcatggatcgttg

acagacagtgggcccaggatctgacattgccatggcaatccggatccggaggcgtt

tggcgcgaaatgcatcatctagtcgagttcgaaccgccacatgccgctacaatcag

agtgttggccctaggcaatcaggagggatcccttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagactgacataccaatggcataaagag

ggatcgtcaatcggtaagttgtttacacagactatgaaaggggtggagagattggc

cgttatgggcgataccgcttgggactttagttccgccggagggttttttactagcg

tcggaaaggggatacataccgtattcggatccgcttttcaggggttgttcggcgga

ctgaattggattacgaaagtgattatgggcgccgtacttatttgggtggggattaa

cactaggaatatgactatgtctatgtctatgatactagtcggagtgattatgatgt

ttctgtcattgggcgtaggcgctGATCAAGGATGCGCCATCAACTTTGGCAAGAGA

GAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCTGATGACTGGCTGAA

CAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCT

CT

YF-Env-
ATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGATGGTTCGTGAG
15

DDDW
GAACCCCTTTTTTGCAGTGACGGCTCTGACCATTGCCTACCTTGTGGGAAGCAACA

E gene
TGACGCAACGAGTCGTGATTGCCCTACTGGTCTTGGCTGTTGGTCCGGCCTACTCA

within this
GCTCACTGCATTGGAATTACTGACAGGGATtttatcgagggggtgcatggcggaac

sequence
ttgggttagcgctacactcgaacaggacaaatgcgttaccgttatggcccccgata

agcctagcctagacattagtctcgaaaccgttgcgatcgatagacccgccgaagtg

agaaaagtgtgttataacgccgtactgactcacgttaagattaacgacaaatgccc

tagtacaggcgaagcgcatctagccgaagagaacgagggcgataacgcatgcaaac

gtacttatagcgatagggggtgggggaacggatgcggattgttcggtaaggggtca

atcgtcgcatgcgctaagtttacatgcgctaagtctatgtcattgttcgaagtcga

tcagactaagattcagtacgtgattagagcgcaattgcatgtgggagcgaaacagg

agaattggactactgacattaagacactgaaattcgacgcccttagcggatcacag

gaggtcgagtttattgggtacggaaaagcgacactcgagtgtcaggtgcagactgc

cgttgactttggcaattcatacatagccgaaatggagacagagtcatggatcgttg

acagacagtgggcccaggatctgacattgccatggcaatccggatccggaggcgtt

tggcgcgaaatgcatcatctagtcgagttcgaaccgccacatgccgctacaatcag

agtgttggccctaggcaatcaggagggatcccttaaaaccgcattgactggcgcta

tgcgcgttactaaggacactaacgacaataacctatacaaactgcatggggggcat

gtgtcttgtagagtgaaattgtccgcccttacacttaaggggactagctataagat

atgcactgacaaaatgtttttcgttaaaaaccctaccgataccggacacggaacag

tcgttatgcaggtgaaagtgtcaaaaggcgcaccatgtaggatacccgtaatcgtt

gccgacgatctgactgccgcaatcaataaggggatactcgtgacagtgaaccctat

cgctagcactaacgacgacgaagtgttgatcgaagtgaatccaccttttggcgact

catacattatcgtaggcagaggcgatagtagaCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGGGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCGGATCAAGGATGCGCCATCAACTTTGGCAAGAGA

GAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCTGATGACTGGCTGAA

CAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCT

CT

YF-Env-
ATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGATGGTTCGTGAG
16

Wt
GAACCCCTTTTTTGCAGTGACGGCTCTGACCATTGCCTACCTTGTGGGAAGCAACA

E gene
TGACGCAACGAGTCGTGATTGCCCTACTGGTCTTGGCTGTTGGTCCGGCCTACTCA

within this
GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAAC

sequence
TTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGACA

AGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGTG

AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCC

CAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCAAGC

GCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAAGGGAGC

ATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTGAGGTTGA

TCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGCCAAGCAGG

AAAATTGGACTACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAGGCTCCCAG

GAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGC

GGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGG

ACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTG

TGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAG

AGTACTGGCCCTGGGAAACCAGGAAGGCTCCTTGAAAACAGCTCTTACTGGCGCAA

TGAGGGTTACAAAGGACACAAATGACAACAACCTTTACAAACTACATGGTGGACAT

GTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCAAGGGGACATCCTACAAAAT

ATGCACTGACAAAATGTTTTTTGTCAAGAACCCAACTGACACTGGCCATGGCACTG

TTGTGATGCAGGTGAAAGTGTCAAAAGGAGCCCCCTGCAGGATTCCAGTGATAGTA

GCTGATGATCTTACAGCGGCAATCAATAAAGGCATTTTGGTTACAGTTAACCCCAT

CGCCTCAACCAATGATGATGAAGTGCTGATTGAGGTGAACCCACCTTTTGGAGACA

GCTACATTATCGTTGGGAGAGGAGATTCACGTCTCACTTACCAGTGGCACAAAGAG

GGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGCCTGGC

CGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGG

TTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTCAGGGGCTATTTGGCGGC

TTGAACTGGATAACAAAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAA

CACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGT

TTTTGTCTCTAGGAGTTGGGGCGGATCAAGGATGCGCCATCAACTTTGGCAAGAGA

GAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCTGATGACTGGCTGAA

CAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCT

CT

Full length genome sequences: 1-118=5′-NTR; 119-481=C (363 nts, 121AAs); 749-973=M (225 nts, 75AAs); 974-2452=E (1479 nts, 493AAs); 2453-3679=NS1 (1227 nts, 409AAs); 3680-4180=NS2a (501 nts, 167AAs); 4181-4570=NS2b (390 nts, 130AAs); 4571-6439=NS3 (1869 nts, 623AAs); 6440-7300=NS4a (861 nts, 287AAs); 7301-7636=NS4b (336 nts, 112AAs); 7637-10354=NS5 (2718 nts, 906AAs); 10355-10862=3′-NTR (508 nts).

The deoptimized YFV, wherein the E protein coding sequence is deoptimized of this invention, is useful in prophylactic and therapeutic compositions for reducing tumor size and treating malignant tumors in various organs, such as: breast, colon, bronchial passage, epithelial lining of the gastrointestinal, upper respiratory and genito-urinary tracts, liver, prostate, the brain, or any other human tissue. In various embodiments, the deoptimized YFV wherein the E protein coding sequence is deoptimized of the present invention are useful for reducing the size of solid tumors and treating solid tumors. In particular embodiments, the tumors treated or reduced in size is glioma, glioblastoma, adenocarcinoma, melanoma, or neuroblastoma. In various embodiments, the tumor is a triple-negative breast cancer.

The pharmaceutical compositions of this invention may further comprise other therapeutics for the prophylaxis of malignant tumors. For example, the deoptimized YFV wherein the E protein coding sequence is deoptimized of this invention may be used in combination with surgery, radiation therapy and/or chemotherapy. Furthermore, one or more deoptimized YFV wherein the E protein coding sequence is deoptimized may be used in combination with two or more of the foregoing therapeutic procedures. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or adverse effects associated with the various monotherapies.

The pharmaceutical compositions of this invention comprise a therapeutically effective amount of one or more deoptimized YFV according to this invention, and a pharmaceutically acceptable carrier. By “therapeutically effective amount” is meant an amount capable of causing lysis of the cancer cells to cause tumor necrosis. By “pharmaceutically acceptable carrier” is meant a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered.

Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the deoptimized viral chimeras.

The compositions of this invention may be in a variety of forms. These include, for example, liquid dosage forms, such as liquid solutions, dispersions or suspensions, injectable and infusible solutions. The preferred form depends on the intended mode of administration and prophylactic or therapeutic application. The preferred compositions are in the form of injectable or infusible solutions.

Methods of Generating Deoptimized YFV Genome, Deoptimized Infectious YF RNA, Deoptimized YF Virus

In various embodiments, the deoptimized YFV of the present invention can be synthesized by well-known recombinant DNA techniques. Any standard manual on DNA technology provides detailed protocols to produce the deoptimized viral chimeras of the invention.

This invention further provides a method of synthesizing any of the viruses described herein, the method comprising (a) identifying the target virus to be synthesized, (b) completely sequencing the target virus or locating the sequence on a publicly or privately available database, (c) de novo synthesis of DNA containing the coding and noncoding region of the genome as a complete plasmid known as an “infectious clone” or as individual pieces of synthetic DNA that can be joined using overlapping PCR. In further embodiments, the entire genome is substituted with the synthesized DNA. In still further embodiments, a portion of the genome is substituted with the synthesized DNA. In yet other embodiments, said portion of the genome is the capsid coding region.

In various embodiments, deoptimized YFV of the present invention is made by first generating a deoptimized viral genome, comprising performing reverse transcription polymerase chain reaction (“RT-PCR”) on a viral RNA from Yellow Fever Virus (YFV) to generate cDNA; performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from the cDNA, wherein the two or more overlapping cDNA fragments collectively encode the YFV; substituting one or more overlapping cDNA fragments comprising a deoptimized sequence for one or more corresponding overlapping cDNA fragment generated from the viral RNA; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences.

In other embodiments, deoptimized YFV of the present invention is made by first generating a deoptimized viral genome, comprising performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from cDNA encoding viral RNA from a YFV, wherein the two or more overlapping cDNA fragments collectively encode the YFV, wherein one or more overlapping cDNA fragments comprises a deoptimized sequence; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences.

In other embodiments, deoptimized YFV of the present invention is made by first generating a deoptimized viral genome, comprising performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from cDNA encoding viral RNA from a YFV, wherein the two or more overlapping cDNA fragments collectively encode the YFV; substituting one or more overlapping cDNA fragments comprising a deoptimized sequence for one or more corresponding overlapping cDNA fragment generated from the viral RNA; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences.

In various embodiments, the method further comprises extracting the viral RNA from the RNA virus prior to performing RT-PCR.

In various embodiments, the deoptimized sequences comprises (1) a recoded sequence having reduced codon pair bias compared to a corresponding sequence on the cDNA, (2) an increased number of CpG or UpA di-nucleotides compared to a corresponding sequence on the cDNA; or (3) at least 5 codons substituted with synonymous codons less frequently used, as discussed herein.

In various embodiments, performing PCR to generate and amplify two or more overlapping cDNA fragments from the cDNA comprises using two or more primer pairs, each pair specific for each of the overlapping cDNA fragments. In various embodiments, performing PCR to generate and amplify two or more overlapping cDNA fragments from the cDNA comprises using two or more primer pairs selected from Table 2.

In various embodiments, the length of the primers is about 15-55 base pairs (bp) in length. In various embodiments, the length of the primers is about 20-40 bp in length. In various embodiments, the length of the primers is about 20-30 bp in length. In various embodiments, the length of the primers is about 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, or 61-65 bp in length.

In various embodiments, performing PCR to generate and amplify two or more overlapping cDNA fragments from the cDNA comprises using 5 or more primer pairs, each pair specific for each of the overlapping cDNA fragments. In various embodiments, the two or more overlapping cDNA fragments from the cDNA is 5 or more overlapping cDNA fragments and the 5 or more overlapping cDNA fragments collectively encode the RNA virus. In various embodiments, performing PCR to generate and amplify 5 or more overlapping cDNA fragments from the cDNA comprises using 5 or more primer pairs selected from Table 2.

In various embodiments, performing PCR to generate and amplify two or more overlapping cDNA fragments from the cDNA comprises using 8 or more primer pairs, each pair specific for each of the overlapping cDNA fragments. In various embodiments, the two or more overlapping cDNA fragments from the cDNA is 8 or more overlapping cDNA fragments and the 8 or more overlapping cDNA fragments collectively encode the RNA virus. In various embodiments, performing PCR to generate and amplify 8 or more overlapping cDNA fragments from the cDNA comprises using 8 or more primer pairs selected from Table 2.

In various embodiments, performing PCR to generate and amplify two or more overlapping cDNA fragments from the cDNA comprises using 10 or more primer pairs, each pair specific for each of the overlapping cDNA fragments. In various embodiments, the two or more overlapping cDNA fragments from the cDNA is 10 or more overlapping cDNA fragments and the 10 or more overlapping cDNA fragments collectively encode the RNA virus.

In various embodiments, performing PCR to generate and amplify two or more overlapping cDNA fragments from the cDNA comprises using 15 or more primer pairs, each pair specific for each of the overlapping cDNA fragments. In various embodiments, the two or more overlapping cDNA fragments from the cDNA is 15 or more overlapping cDNA fragments and the 15 or more overlapping cDNA fragments collectively encode the RNA virus.

In various embodiments, performing PCR to generate and amplify two or more overlapping cDNA fragments from the cDNA comprises using two or more primer pairs selected from Table 2.

TABLE 2

SEQ

ID
Primer Usage/PCR

Primer #
Primer Sequence
NO:
Product Size

2557- YFVF1 F
AGTAAATCCTGTGTGCTAATTGAGGTG
17
for YFV Fragment 1

(998 bp)

2520-YFVF1-R
TGTCAGTAATTCCAATGCAGTGAG
18
for YFV Fragment 1

(998 bp)

2519-YFVF1-F
AGCTTATCATCGATAAGCTTGCTAGC
19
for YFV Fragment 1

containing phi2.5 T7

promoter (1046 bp)

2520-YFVF1-R
TGTCAGTAATTCCAATGCAGTGAG
20
for YFV Fragment 1

containing phi2.5 T7

promoter (1046 bp)

2521-YFVF2-F
ATGACTGGAAGAATGGGTGAAAGG
21
for YFV Fragment 2

(1794 bp)

2522-YFVF2-R
AGAGGCTTTCACTATTGATGCAAGC
22
for YFV Fragment 2

(1794 bp)

2523-YFVF3-F
ATCAAGGATGCGCCATCAACTTTG
23
for YFV Fragment 3

(1550 bp)

2524-YFVF3-R
AAGTCTCACCTCAGCCATAGTGAC
24
for YFV Fragment 3

(1550 bp)

2525-YFVF4-F
AACGCCTTGTGCTGACCCTAG
25
for YFV Fragment 4

(1596 bp)

2526-YFVF4-R
TTGGTTCCAACATCCTGTAAGTTAG
26
for YFV Fragment 4

(1596 bp)

2527-YFVF5-F
ATCTTGGCCGAGTGCGCACG
27
for YFV Fragment 5

(1598 bp)

2528-YFVF5-R
TCGGGGATCACAACCACCATC
28
for YFV Fragment 5

(1598 bp)

2529-YFVF6-F
TGCTGTTTATACTGGCTGGACTAC
29
for YFV Fragment 6

(1601 bp)

2530-YFVF6-R
TGGCATGTATGGAGCTAACACC
30
for YFV Fragment 6

(1601 bp)

2531-YFVF7-F
ATCATCACCTTCAAGGACAAAACTG
31
for YFV Fragment 7

(1600 bp)

2532-YFVF7-R
ATCCGTGCTCAGTGAGCCATG
32
for YFV Fragment 7

(1600 bp)

2533-YFVF8-F
AGCCTACATGGATGTCATAAGTC
33
for YFV Fragment 8

(1460 bp)

2534-YFVF8-R
AGTGGTTTTGTGTTTGTCATCCAAAG
34
for YFV Fragment 8

(1460 bp)

2862-YFVF5 F
ACG TGA AAT TCC ACA CAC AGG C
35
For YFV Fragment 5

(1537 bp)

2863-YFVF5_R
ACT TGG TTG TCT TGG ATG GAC C
36
For YFV Fragment 5

(1537 bp)

2529-YFVF6_F
TGC TGT TTA TAC TGG CTG GAC TAC
37
For YFV Fragment 6

(1601 bp)

2530-YFVF6 R
TGG CAT GTA TGG AGC TAA CAC C
38
For YFV Fragment 6

(1601 bp)

2531-YFVF7 F
ATCATCACCTTCAAGGACAAAACT G
39
For YFV Fragment 7

(1606 bp)

2864-YFVF7 R
TGT TAC ATC CGT GCT CAG TGA G
40
For YFV Fragment 7

(1606 bp)

2533-YFVF8 F
AGC CTA CAT GGA TGT CAT AAG TC
41
For YFV Fragment 8

(1460 bp)

2865-YFVF8_R
AGT GGT TTT GTG TTT GTC ATC CAA AGG
42
For YFV Fragment 8

TCT GC

(1460 bp)

In various embodiments, the length of the overlap is about 40-400 bp. In various embodiments, the length of the overlap is about 200 bp. In various embodiments, the length of the overlap is about 40-100 bp. In various embodiments, the length of the overlap is about 100-200 bp. In various embodiments, the length of the overlap is about 100-150 bp. In various embodiments, the length of the overlap is about 150-200 bp. In various embodiments, the length of the overlap is about 200-250 bp. In various embodiments, the length of the overlap is about 200-300 bp. In various embodiments, the length of the overlap is about 300-400 bp.

In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence having one or more mutations relative to a corresponding sequence on the cDNA. In certain embodiments, there are 5 or more mutations. In certain embodiments, there are 10 or more mutations. In certain embodiments, there are 20 or more mutations. In certain embodiments there are 50 or more mutations. In certain embodiments there are 100 or more mutations. The one or more mutations can be a deletion, addition, substitution or combinations thereof.

In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence having up to 2% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA. In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence that results in having up to 1.75% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA. In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence having up to 1.5% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA. In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence having up to 1.25% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA. In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence having up to 1% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA. In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence having up to 0.75% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA. In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence having up to 0.5% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA. In various embodiments, each of the one or more overlapping cDNA fragments comprising the deoptimized sequence comprises a sequence encoding an amino acid sequence that having up to 0.25% amino acid substitutions, additions or deletions relative to the amino acid sequence encoded by the corresponding sequence on the cDNA.

In various embodiments, performing overlapping PCR to construct the deoptimized viral genome is done on the two or more overlapping cDNA fragments at the same time. Thus, if there are 5 more overlapping cDNA fragments, overlapping PCR to construct the deoptimized viral genome is done on those 5 fragments at the same time. As further examples, if there are 8 more overlapping cDNA fragments, overlapping PCR to construct the deoptimized viral genome is done on those 8 fragments at the same time; if there are 10 more overlapping cDNA fragments, overlapping PCR to construct the deoptimized viral genome is done on those 10 fragments at the same time.

In various embodiments, the methods do not use an intermediate DNA clone, such as a plasmid, BAC or YAC. In various embodiments, the methods do not use a cloning host. In various embodiments, the methods do not include an artificial intron in the sequences; for example, to disrupt an offending sequence locus.

Various embodiments of the invention provide for a method of generating a deoptimized infectious YFV RNA, comprising: performing in vitro transcription of a deoptimized viral genome to generate a deoptimized RNA transcript.

In various embodiments, the method comprises generating the deoptimized viral genome in accordance with embodiments of the present invention before performing the in vitro transcription.

Thus, in various embodiments, the method comprises performing reverse transcription polymerase chain reaction (“RT-PCR”) on a viral RNA from a Yellow Fever virus to generate cDNA; performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from the cDNA, wherein the two or more overlapping cDNA fragments collectively encode the YF virus; substituting one or more overlapping cDNA fragments comprising a deoptimized sequence for one or more corresponding overlapping cDNA fragment generated from the viral RNA; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences; and performing in vitro transcription of a deoptimized viral genome to generate a deoptimized RNA transcript.

In other embodiments, the method comprises performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from cDNA encoding viral RNA from a YF virus, wherein the two or more overlapping cDNA fragments collectively encode the YF virus, wherein one or more overlapping cDNA fragments comprises a deoptimized sequence; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences; and performing in vitro transcription of a deoptimized viral genome to generate a deoptimized RNA transcript.

In other embodiments, the method comprises performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from cDNA encoding viral RNA from a YF virus, wherein the two or more overlapping cDNA fragments collectively encode the YF virus; substituting one or more overlapping cDNA fragments comprising a deoptimized sequence for one or more corresponding overlapping cDNA fragment generated from the viral RNA; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences; and performing in vitro transcription of a deoptimized viral genome to generate a deoptimized RNA transcript.

In various embodiments, the method further comprising extracting the viral RNA from the YF virus prior to performing RT-PCR.

Specific embodiments of the deoptimized viral genome and methods of generating the deoptimized viral genome are as provided above and below and are included in these embodiments for generating deoptimized infectious YFV RNA.

Various embodiments of the invention provide for a method of generating a deoptimized YF virus, comprising transfecting host cells with a quantity of a deoptimized infectious RNA; culturing the host cells; and collecting infection medium comprising the deoptimized virus.

In various embodiments, the method comprises performing reverse transcription polymerase chain reaction (“RT-PCR”) on a viral RNA from a Yellow Fever virus to generate cDNA; performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from the cDNA, wherein the two or more overlapping cDNA fragments collectively encode the YF virus; substituting one or more overlapping cDNA fragments comprising a deoptimized sequence for one or more corresponding overlapping cDNA fragment generated from the viral RNA; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences; performing in vitro transcription of a deoptimized viral genome to generate a deoptimized RNA transcript; culturing the host cells; and collecting infection medium comprising the deoptimized virus.

In various embodiments, the method further comprises generating the quantity of deoptimized infectious RNA in accordance with various embodiments of the present invention before transfecting host cells with the quantity of the deoptimized infectious RNA. Thus, the invention comprises performing in vitro transcription of a deoptimized viral genome to generate a deoptimized RNA transcript; and transfecting host cells with a quantity of a deoptimized infectious RNA; culturing the host cells; and collecting infection medium comprising the deoptimized virus.

In other embodiments, the method comprises performing polymerase chain reaction (“PCR”) to generate and amplify two or more overlapping cDNA fragments from cDNA encoding viral RNA from a YF virus, wherein the two or more overlapping cDNA fragments collectively encode the YF virus; substituting one or more overlapping cDNA fragments comprising a deoptimized sequence for one or more corresponding overlapping cDNA fragment generated from the viral RNA; performing overlapping and amplifying PCR to construct the deoptimized viral genome, wherein the deoptimized viral genome comprises one or more deoptimized sequences; and performing in vitro transcription of a deoptimized viral genome to generate a deoptimized RNA transcript; culturing the host cells; and collecting infection medium comprising the deoptimized virus.

In various embodiments, the method further comprising extracting the viral RNA from the RNA virus prior to performing RT-PCR.

Specific embodiments of the deoptimized viral genome, methods of generating the deoptimized viral genome, and the infectious YFV RNA and generating the infectious YFV RNA are as provided above and below and are included in these embodiments for generating deoptimized YFV.

Example of host cells include, but are not limited to Vero E6 cells, MDCK cells, HeLa cells, Chicken embryo fibroblasts, embryonated chicken eggs, MRC-5 cells, WISTAR cells, PERC.6 cells, Huh-7 cells, BHK cells, MA-10⁴cells, Vero cells, WI-38 cells, and HEK 293 cells.

Immune and or Vaccines Compositions

Various embodiments provide for an immune composition for inducing an immune response in a subject, comprising: a deoptimized Yellow Fever Virus of the present invention. The deoptimized Yellow Fever Virus is any one of the deoptimized Yellow Fever Virus discussed herein. In various embodiments, the deoptimized Yellow Fever Virus of the present invention is a live-attenuated virus. In some embodiments the immune composition further comprises an acceptable excipient or carrier as described herein. In some embodiments, the immune composition further comprises a stabilizer as described herein. In some embodiments, the immune composition further comprise an adjuvant as described herein. In some embodiments, the immune composition further comprises sucrose, glycine or both. In various embodiments, the immune composition further comprises about sucrose (5%) and about glycine (5%). In various embodiments, the acceptable carrier or excipient is selected from the group consisting of a sugar, amino acid, surfactant and combinations thereof. In various embodiments, the amino acid is at a concentration of about 5% w/v. Nonlimiting examples of suitable amino acids include arginine and histidine. Nonlimiting examples of suitable carriers include gelatin and human serum albumin. Nonlimiting examples of suitable surfactants include nonionic surfactants such as Polysorbate 80 at very low concentration of 0.01-0.05%.

In various embodiments, the immune composition is provided at dosages of about 10³-10⁷PFU. In various embodiments, the immune composition is provided at dosages of about 10⁴-10⁶PFU. In various embodiments, the immune composition is provided at a dosage of about 10³PFU. In various embodiments, the immune composition is provided at a dosage of about 10⁴PFU. In various embodiments, the immune composition is provided at a dosage of about 10⁵PFU. In various embodiments, the immune composition is provided at a dosage of about 10⁶PFU. In various embodiments, the immune composition is provided at a dosage of about 10⁷PFU. In various embodiments, the immune composition is provided at a dosage of about 10⁸PFU.

In various embodiments, the immune composition is provided at a dosage of about 5×10³PFU. In various embodiments, the immune composition is provided at a dosage of about 5×10⁴PFU. In various embodiments, the immune composition is provided at a dosage of about 5×10⁵PFU. In various embodiments, the immune composition is provided at a dosage of about 5×10⁶PFU. In various embodiments, the immune composition is provided at a dosage of about 5×10⁷PFU. In various embodiments, the immune composition is provided at a dosage of about 5×10⁸PFU.

In various embodiments, the immune composition is provided at a dosage of about 3×10⁴PFU. In various embodiments, the immune composition is provided at a dosage of about 3×10⁵PFU. In various embodiments, the immune composition is provided at a dosage of about 3×10⁶PFU. In various embodiments, the immune composition is provided at a dosage of about 3×10⁷PFU. In various embodiments, the immune composition is provided at a dosage of about 3×10⁸PFU.

In various embodiments, the immune composition is provided at a dosage of about 6.25×10⁵PFU. In various embodiments, the immune composition is provided at a dosage of about 6.25×10⁶PFU. In various embodiments, the immune composition is provided at a dosage of about 6.25×10⁷PFU. In various embodiments, the immune composition is provided at a dosage of about 6.25×10⁸PFU. In various embodiments, the immune composition is provided at a dosage of about 6.25×10⁹PFU.

Various embodiments provide for a vaccine composition for inducing an immune response in a subject, comprising: a deoptimized Yellow Fever Virus of the present invention. The deoptimized Yellow Fever Virus is any one of the deoptimized Yellow Fever Virus discussed herein. In various embodiments, the deoptimized Yellow Fever Virus of the present invention is a live-attenuated virus. In some embodiments the vaccine composition further comprises an acceptable carrier or excipient as described herein. In some embodiments, the immune composition further comprises a stabilizer as described herein. In some embodiments, the vaccine composition further comprise an adjuvant as described herein. In some embodiments, the vaccine composition further comprises sucrose, glycine or both. In various embodiments, the vaccine composition further comprises sucrose (5%) and glycine (5%). In various embodiments, the acceptable carrier or excipient is selected from the group consisting of a sugar, amino acid, surfactant and combinations thereof. In various embodiments, the amino acid is at a concentration of about 5% w/v. Nonlimiting examples of suitable amino acids include arginine and histidine. Nonlimiting examples of suitable carriers include gelatin and human serum albumin. Nonlimiting examples of suitable surfactants include nonionic surfactants such as Polysorbate 80 at very low concentration of 0.01-0.05%.

In various embodiments, the vaccine composition is provided at dosages of about 10³-10⁷PFU. In various embodiments, the vaccine composition is provided at dosages of about 10⁴-10⁶PFU. In various embodiments, the vaccine composition is provided at a dosage of about 10³PFU. In various embodiments, the vaccine composition is provided at a dosage of about 10⁴PFU. In various embodiments, the vaccine composition is provided at a dosage of about 10⁵PFU. In various embodiments, the vaccine composition is provided at a dosage of about 10⁶PFU. In various embodiments, the vaccine composition is provided at a dosage of about 10⁷PFU. In various embodiments, the vaccine composition is provided at a dosage of about 10⁸PFU. In various embodiments, the vaccine composition is provided at a dosage of about 10⁹PFU.

In various embodiments, the vaccine composition is provided at a dosage of about 5×10³PFU. In various embodiments, the vaccine composition is provided at a dosage of about 5×10⁴PFU. In various embodiments, the vaccine composition is provided at a dosage of about 5×10⁵PFU. In various embodiments, the vaccine composition is provided at a dosage of about 5×10⁶PFU. In various embodiments, the vaccine composition is provided at a dosage of about 5×10⁷PFU.

In various embodiments, the vaccine composition is provided at a dosage of about 3×10⁴PFU. In various embodiments, the vaccine composition is provided at a dosage of about 3×10⁵PFU. In various embodiments, the vaccine composition is provided at a dosage of about 3×10⁶PFU. In various embodiments, the vaccine composition is provided at a dosage of about 3×10⁷PFU.

In various embodiments, the vaccine composition is provided at a dosage of about 6.25×10⁵PFU. In various embodiments, the vaccine composition is provided at a dosage of about 6.25×10⁶PFU. In various embodiments, the vaccine composition is provided at a dosage of about 6.25×10⁷PFU. In various embodiments, the vaccine composition is provided at a dosage of about 6.25×10⁸PFU. In various embodiments, the vaccine composition is provided at a dosage of about 6.25×10⁹PFU.

Various embodiments provide for a vaccine composition for inducing a protective immune response in a subject, comprising: a deoptimized Yellow Fever Virus of the present invention. The deoptimized Yellow Fever Virus is any one of the deoptimized Yellow Fever Virus discussed herein. In various embodiments, the deoptimized Yellow Fever Virus of the present invention is a live-attenuated virus. In some embodiments the vaccine composition further comprises an acceptable carrier or excipient as described herein. In some embodiments, the vaccine composition further comprise an adjuvant as described herein. In some embodiments, the vaccine composition further comprises sucrose, glycine or both. In various embodiments, the vaccine composition further comprises sucrose (5%) and glycine (5%). In various embodiments, the acceptable carrier or excipient is selected from the group consisting of a sugar, amino acid, surfactant and combinations thereof. In various embodiments, the amino acid is at a concentration of about 5% w/v. Nonlimiting examples of suitable amino acids include arginine and histidine. Nonlimiting examples of suitable carriers include gelatin and human serum albumin. Nonlimiting examples of suitable surfactants include nonionic surfactants such as Polysorbate 80 at very low concentration of 0.01-0.05%.

It should be understood that an attenuated virus of the invention, where used to elicit an immune response in a subject (or protective immune response) or to prevent a subject from or reduce the likelihood of becoming afflicted with a virus-associated disease, can be administered to the subject in the form of a composition additionally comprising a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers and excipients are known to those skilled in the art and include, but are not limited to, one or more of 0.01-0.1M and preferably 0.05M phosphate buffer, phosphate-buffered saline (PBS), DMEM, L-15, a 10-25% sucrose solution in PBS, a 10-25% sucrose solution in DMEM, or 0.9% saline. Such carriers also include aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Solid compositions may comprise nontoxic solid carriers such as, for example, glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate, gelatin, recombinant human serum albumin, human serum albumin, and/or magnesium carbonate. For administration in an aerosol, such as for pulmonary and/or intranasal delivery, an agent or composition is preferably formulated with a nontoxic surfactant, for example, esters or partial esters of C6 to C22 fatty acids or natural glycerides, and a propellant. Additional carriers such as lecithin may be included to facilitate intranasal delivery. Pharmaceutically acceptable carriers or excipients can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients. The instant compositions can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject.

In various embodiments, the vaccine composition or immune composition is formulated for delivery intravenously, or intrathecally, subcutaneously, intramuscularly, intradermally or intranasally. In various embodiments, the vaccine composition or immune composition is formulated for delivery intranasally. In various embodiments, the vaccine composition or immune composition is formulated for delivery via a nasal drop or nasal spray.

As discussed, any one of the deoptimized Yellow Fever Virus of the present invention can be used in the immune compositions or vaccine compositions discussed herein.