Attenuated virus having multiple hosts

This application claims priority to U.S. Application No. 62/046,565 filed Sep. 5, 2014, and U.S. Application No. 62/050,638 filed Sep. 15, 2014, which are incorporated herein by reference in their entireties.

This invention was made with government support under Grant Nos. AI07521901 and GM098400 awarded by the National Institute of Health. The government has certain rights in the invention.

A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

The present invention provides attenuated viruses, methods for making attenuated viruses, and vaccine compositions comprising one or more attenuated viruses, wherein the attenuated virus comprises a modified viral genome containing a plurality of nucleotide substitutions that result in the rearrangement of codons of one or more virus protein encoding sequences and changes in codon pair bias compared to one or more viral hosts. The attenuated viruses enable production of improved vaccines and are used to elicit protective immune response.

Viruses that infect multiple phylogenetically distant hosts, for example hosts from different kingdoms, phyla, or classes, must accommodate the differences in the hosts so that the virus can efficiently replicate in hosts with different biochemical and molecular processes. These host differences include, for example, thermoregulation, protein glycosylation patterns, biochemical features of cell membranes, and CpG dinucleotide frequencies. Arboviruses, for example, have the ability to infect vertebrate and arthropod hosts.

The term arbovirus ( ar thropod- bo rne virus) applies to any virus that is transmitted to humans and/or other vertebrates by certain species of blood-feeding arthropods, chiefly insects (flies and mosquitoes) and arachnids (ticks). Families in the current classification system that have some arbovirus members include Bunyaviridae (comprising the bunyaviruses, phleboviruses, nairoviruses, and hantaviruses), Flaviviridae (comprising only the flaviviruses), Reoviridae (comprising the coltiviruses and orbiviruses), and Togaviridae (comprising the alphaviruses). Birds are often reservoirs for arboviruses, which are transmitted by mosquitoes to horses, other domestic animals, and humans. Certain arboviruses are transmissible by humans, including dengue fever, yellow fever, and chikungunya disease, which can be transmitted from person to person via mosquitoes.

Dengue virus (DENV) is an enveloped, plus stranded RNA arbovirus (genome ˜11 kb) of the genus Flavivirus of the Flaviviridae family. DENV is primarily transmitted by the urban-adapted Aedes aegypti mosquito vector that has become widely distributed in tropical and subtropical regions. The diseases resulting from DENV infection include self-limiting dengue fever (DF), life-threatening dengue shock syndrome (DSS), and dengue hemorrhagic fever (DHF) characterized by increased vascular permeability and thrombo-cytopenia. DENV infections are one of the leading causes of arthropod-borne human diseases in the world. Each year there is an estimated 50-200 million DENV infections world-wide, resulting in 500,000 cases of DHF/DSS, and over 20,000 deaths, with 3.6 billion people at risk.

There are five antigenically distinct serotypes of DENV. Infection with one serotype induces immunity against that serotype and some degree of cross-protection against the other serotypes. However, the cross-protective immunity typically persists only for a relatively short time. Also, cross-reactive antibodies may bind to, but not neutralize other serotypes, leading to more severe secondary infections. An effective dengue vaccine would preferably be protective against all known serotypes. Currently, there are no marketable vaccines available capable of preventing human infection by any of the DENV serotypes.

Codon pair preference, or codon pair bias, refers to a phenomenon in which certain pairs of adjacent codons are used more frequently or less frequently in a particular host than expected after accounting for the frequency of usage of the individual codons (Gutman & Hatfield, 1989; Moura et al., 2007; Coleman et al., 2008). Every codon pair can be assigned a codon pair score (CPS), which is the natural logarithm of the ratio of the observed frequency of the codon pair to the expected frequency of the codon pair (i.e., CPS=ln(Observed/Expected) (Coleman et al., 2008).

In one aspect the invention provides an attenuated virus containing a viral genome having one or more modified virus protein-encoding sequences wherein the codon pair bias, relative to a first host, of at least one virus protein-encoding sequence is less than the codon pair bias of the parent nucleic acid sequence from which it is derived, and wherein the codon pair bias of the one or more modified virus protein-encoding sequences is not substantially reduced relative to that of a second host. In one embodiment, the codon pair bias of the one or more modified virus protein-encoding sequences in the attenuated virus is reduced relative to the first host by at least 0.05, at least 0.1, at least 0.2, at least 0.3, or at least 0.4. In a further embodiment, the codon pair bias of the one or more modified virus protein-encoding sequences is within 0.002, 0.005, 0.010, 0.020, or 0.050 of the parent nucleic acid from which it is derived relative to the second host. In one embodiment, the codon pair bias of the one or more modified virus protein-encoding sequences is reduced relative to the first host by codon rearrangement of the parent nucleic acid without substantially changing the codon usage.

In another aspect, the invention provides an attenuated virus comprising a viral genome having one or more modified virus protein-encoding sequences wherein the codon pair bias, relative to a first host and a second host, of at least one virus protein-encoding sequence is less than the codon pair bias of the parent nucleic acid from which it is derived. In one embodiment, the codon pair bias of the one or more modified virus protein-encoding sequences is reduced relative to the first host and second host independently by at least 0.05, at least 0.1, at least 0.2, at least 0.3, or at least 0.4.

In one aspect, the invention provides a method of making an attenuated virus genome comprising the steps: (a) obtaining an virus protein-encoding sequence; (b) rearranging synonymous codons of the protein-encoding sequence to obtain a modified protein-encoding sequence that (i) encodes the same amino acid sequence as the unrearranged protein-encoding sequence, (ii) has a reduced codon pair bias relative to a first host compared to the protein-encoding nucleotide sequence, (iii) has a substantially similar codon pair bias relative to a second host compared to the unrearranged nucleotide sequence; and (c) substituting all or part of the modified nucleotide sequence into the unrearranged genome of a parent virus. In one embodiment, the codon pair bias of the modified protein encoding sequence relative to the first host is reduced by at least 0.05, at least 0.1, at least 0.2, at least 0.3, or at least 0.4 compared to the unrearranged protein encoding nucleotide sequence. In one embodiment, the codon pair bias of the modified protein encoding sequence relative to the second host is within 0.002, 0.005, 0.010, 0.020, or 0.050 of the unrearranged nucleotide sequence. In one embodiment, an attenuated virus is made by inserting the attenuated viral genome into a cell line.

In another aspect, the invention provides a method of making an attenuated virus genome comprising the steps: (a) obtaining an virus protein-encoding sequence; (b) rearranging synonymous codons of the protein-encoding sequence to obtain a modified protein-encoding sequence that (i) encodes the same amino acid sequence as the unrearranged protein-encoding sequence, (ii) has a reduced codon pair bias relative to a first host compared to the protein-encoding nucleotide sequence, (iii) has a substantially reduced codon pair bias relative to a second host compared to the unrearranged nucleotide sequence; and (c) substituting all or part of the modified nucleotide sequence into the unrearranged genome of a parent virus. In one embodiment, the codon pair bias of the modified protein encoding sequence relative to the first host and second host is independently reduced by at least 0.05, at least 0.1, at least 0.2, at least 0.3, or at least 0.4 compared to the unrearranged protein encoding nucleotide sequence. In one embodiment, an attenuated virus is made by inserting the attenuated viral genome into a cell line.

In one embodiment of the invention, the first host is a vertebrate. In a further embodiment the first host in a mammal. In a further embodiment the first host is a human. In one embodiment, the second host is an arthropod. In further embodiment the second host is an arachnid. In one embodiment the second host is a tick. In one embodiment the second host is an insect. In one embodiment the second host is a mosquito.

In one embodiment, the virus is attenuated in the first host, but replicates efficiently in the second host and cell lines derived from the second host. In one embodiment, the codon pair bias of the one or more modified virus protein-encoding sequences is increased relative to the second host.

In one embodiment, the attenuated virus is an arbovirus. In a further embodiment, the attenuated arbovirus is selected from the group consisting of Bunyaviridae (comprising the bunyaviruses, phleboviruses, nairoviruses, and hantaviruses), Flaviviridae (comprising only the flaviviruses), Reoviridae (comprising the coltiviruses and orbiviruses), and Togaviridae (comprising the alphaviruses). In one embodiment, the attenuated virus is a flavavirus. In one embodiment, the attenuated virus is a dengue virus.

In one embodiment, the one or more modified virus protein-encoding sequences is derived from the nucleic acid sequence encoding the dengue virus protein-encoding sequence, or a portion thereof, selected from one or more of the group consisting of C; prM; E; NS1; 2A; 2B; NS3; 4A; 4B, and NS5. In one embodiment, the modified virus protein-encoding sequence is derived from the nucleic acid sequence encoding the E structural glycoprotein. In one embodiment, the modified virus protein-encoding sequence is derived from the nucleic acid sequence encoding the NS3 multi-functional protease. In one embodiment, the modified virus protein-encoding sequence is derived from the nucleic acid sequence encoding the NS5 multifunctional RNA polymerase.

In one aspect, the invention provides a vaccine composition for inducing a protective immune response in a subject, wherein the vaccine composition comprises an attenuated virus described herein. In one embodiment, the vaccine composition induces a protective immune response in a subject comprising an attenuated arbovirus described herein, wherein the protective immune response is against one or more Dengue virus serotypes selected from the group consisting of dengue virus serotypes 1 to 5. In one aspect the invention provides a method of eliciting a protective immune response in a subject comprising administering to the subject a prophylactically or therapeutically effective dose of the vaccine composition comprising an attenuated virus as described herein.

FIG. 1 . Construction of a synthetic wild-type dengue serotype 2 virus. (A) A synthetic wild-type DENV-2 virus was designed based on strain 16681 genome (Accession# U87411) and divided into four fragments incorporating 26 silent mutations (listed in Table 4). Fragment 1 contained an upstream T7 promoter with no extraneous 5′ G nt inserted between the T7 promoter and the 5′-terminal A nt of the genomic cDNA. Each fragment was designed with different sets of restriction sites at the 5′ and 3′ ends to facilitate ligation, synthesized by GenScript, and placed into a high-copy pUC57 plasmid. (B) To construct full-length DENV-2 genome, each fragment was ligated into the low-copy pBR322 plasmid in the following order using specified restriction enzymes: Fragment 4 (AvaI/SphI), Fragment 3 (ClaI/StuI), Fragment 2 (NheI/KpnI), and Fragment 1 (ClaI/SacI).

FIG. 2 . (A) Growth of synthetic wild-type (D2-syn) compared to DENV strain 16681. Infectivity of RNA transcripts was verified by indirect immunofluorescence using culture media collected from the third or fourth blind passages, 7-9 days post-infection. (B) To verify that the 26 silent mutations did not alter the growth phenotype and kinetics of D2-syn compared to the wild-type 16681 virus, plaque titrations were performed in both C6/36 and LLC-MK2 rhesus monkey kidney cells at a MOI of 0.01. Plaque sizes and phenotype were found to be similar between D2-syn and 16681 viruses. (C) Growth kinetics was also found to be similar, with D2-syn and 16681 reaching maximal titers of 1.3×10 7 PFU/mL and 5.5×10 6 PFU/mL respectively in LLC-MK2 cells at Day 7 and 6×10 7 PFU/mL and 5×10 7 PFU/mL respectively in C6/36 cells at Day 9.

FIG. 3 . Codon pair use by two arthropod vector transmitted viruses infecting either plant or animal primary hosts. (A) Correlation of codon pair use frequency between sheep ( O. aries ) and mosquito ( A. aegypti ) genomes and (B) the frequency at which these codon pairs are used in the Rift Valley fever virus genome. (C) A similar comparison between maize ( Z. mays ) and leafhopper ( G. nigrifronts ) which are host to (D) Maize fine streak virus. Coding DNA sequences for the leafhopper were generated using transcriptome data from NCBI Bioproject PRGNA200322 and the Augustus gene prediction program.

FIG. 4 . Codon pair bias in humans and mosquitoes. (A) Codon pair preferences are well-correlated (Spearman rho=0.95) between humans and mice. (B) Codon pair preferences are poorly correlated (Spearman rho=0.26) between humans and mosquitoes. Each circle represents one of the 3,721 possible codon pairs. (C) Codon pairs actually used by natural wild-type dengue virus, type 2 (16681). The more times a particular codon pair is used by the virus, the bigger and darker the dot. (D) Codon pairs used by an in silico recoded dengue virus designed to have a good codon pair score in mosquitoes but a bad (negative) codon pair score in humans (dots). “hmin” signifies a human minimized virus.

FIG. 5 . (A) The average human codon pair score of the in silico hmin virus (grey dot) compared with WT (black dot) and the average codon pair scores of >14,000 human coding genes. (B) The same two viruses as in E (grey and black dots) evaluated using mosquito codon pair scores and compared with all Aedes aegypti mosquito coding genes. The in silico hmin virus demonstrates that it is possible to design a synthetic dengue virus drastically deoptimized in humans but optimized in mosquitoes.

FIG. 6 . Design and growth kinetics of WT (D2-syn) and three hmin dengue viruses in mammalian and mosquito cell lines. (A) (Top) Diagram of the DENV2 genome marks, the polyprotein coding region and the coding regions of polypeptides before proteolytic processing. The color-coded regions indicate regions recoded in the three novel hmin viruses. Full length genomes of the three hmin viruses (E hmin , NS3 hmin , and NS5 hmin ) are aligned to the WT (D2-syn) sequence, and point mutations generated by codon pair recoding are indicated by a barcode diagram. The two CPS line plots (Bottom) show how the codon pair score changes along the length of the genome for each virus relative to the mosquito and human CPBs. There are four overlapping loess curves: E hmin , NS3 hmin , NS5 hmin , and D2-syn. (B-D) Virus growth curves in different cell lines produced by measuring the fold change in virus RNA concentration from time 0.

FIG. 7 . Growth curves and plaque phenotypes of viruses grown in different cells. (A) Insect C6/36 cells or (B) mammalian LLC-MK2 cells were infected with virus variants at a multiplicity of infection (MOI) of 0.001. Virus titer was measured by plaque assay on BHK cells. (C) NS5 hmin growth kinetics in different cells. C6/36 or LLC-MK2 cells were infected with NS5 hmin at a MOI of 1. Virus titer was measured by plaque assay on BHK cells. (D) BHK cell plaque phenotypes using virus grown in LLC-MK2, or (E) C6/36 cells at 0.01 MOI. (F) Plaque phenotypes were more evident for NS5 hmin using virus grown in C6/36 cells at 1 MOI. (G) Virus titers, measured by focus forming assays in C6/36, BHK-21, Vero E6, A549, or LLC-MK2 cell lines. (H) Effect of Jak inhibitor 1 treatment on virus titer. LLC-MK2 cells were pretreated with Jak inhibitor 1, and the fold change in virus titer relative to untreated cells was measured by a 50% tissue culture infectious dose (TCID 50 ) assay at three and seven days post infection. Significant differences from D2-syn in (G) and (H) are marked by *P value<0.05 by Wilcox rank sum test.

FIG. 8 . Survival curves showing attenuation of the hmin viruses in newborn mice. (A and B) Attenuation of hmin viruses after intracerebral infection. Groups of newborn ICR mice (1-2 d old) were infected intracerebrally with 10 3 (A) or 10 4 PFU (B) of (D2-syn) or hmin viruses, respectively. (C) Median lethal dose (LD 50 ) values in newborn mice after intracerebral infection. (D) Maternal antibody PRNT 50 titer in juvenile mice born to mothers vaccinated (when they were newborn animals) with D2-syn or E hmin (*P value<0.05 by Wilcox rank sum test).

Arboviruses exhibit life cycles that involve both vertebrates and arthropods. In order to infect and replicate in these two very different types of hosts, the virus must be able to adapt to growth conditions that are very different, including temperature, host factors, cell membrane thickness and composition, and even differences in genome synonymous codon usage and codon pair bias. All species have preferences in the ways they encode proteins in nucleic acids and then translate them. Provided the degeneracy of the genetic code, different organisms have evolved diverging preferences for encoding proteins reflected by differences in codon bias (or codon usage) and codon pair bias (CPB). CPB is the preferential use of certain codon pairings to encode adjacent amino acids compared to what would be expected based on the codon usage of each of the two synonymous codons encoding the amino acid pair. WO 08/121992, which is incorporated by reference, provides a description of codon-pair bias.

It has been discovered that CPB diverges dramatically with increasing evolutionary distance such as between mammals and insects. For example, adjacent codon pairs under- or over-represented in humans tend to be under- or over-represented in other mammals, and adjacent codon pairs under- or over-represented in Aedes aegypti (an insect vector for certain arboviruses) tend to be under- or over-represented in certain other insects, but there is little such similarity in codon pair preference between insects and mammals.

The present invention relates to attenuated viruses comprising a viral genome that has been engineered to contain one or more modified virus protein encoding sequences that have a codon pair bias that is less than the nucleic acid sequence from which it was derived relative to a first viral host, while not substantially reducing the codon pair bias of the modified sequence relative to a second host. In this embodiment, the virus is designed to be attenuated in one host while maintaining the virus's ability to grow efficiently in the second host.

In another embodiment, the invention relates to an attenuated virus comprising a viral genome that has been engineered to contain one or more modified virus protein-encoding sequences that have a codon pair bias that is less than the codon pair bias of the parent nucleic acid from which it is derived, relative to a first host and a second host. In this embodiment, the virus is designed to be attenuated in both hosts.

The invention utilizes the differences in codon-pair bias between phylogenetically distant organisms to (i) deoptimize a virus for growth in one host while optimizing (or leaving unchanged) growth in another host; (ii) deoptimize the virus for growth in both hosts; or (iii) optimize the virus for growth in both hosts. In one embodiment of the invention, the differences in codon-pair bias between insects and mammals is used to design and synthesize a live arbovirus (e.g., dengue virus) that is attenuated in a mammalian host, but replicates efficiently in insects and insect-derived cell lines. In another embodiment, an arbovirus is designed that is attenuated in both mammalian and insect hosts. The methods described herein can be applied to make other arboviruses besides dengue, that are attenuated in mammals or other vertebrate hosts, in a phylum-specific manner.

The present invention relates to the production of attenuated virus that can be used in vaccines to protect against viral infection and disease. Accordingly, the invention provides an attenuated virus, which comprises a modified viral genome containing nucleotide substitutions engineered in one or more virus protein encoding sequences, wherein the substitutions introduce a plurality of rearranged synonymous codons into the genome. In one embodiment, the order of existing codons is changed, as compared to a reference (e.g., a wild type) viral sequence, while maintaining the reference amino acid sequence. The change in codon order alters the occurrence of codon pairs, and consequently, alters codon pair bias relative to at least one viral host.

Most amino acids are encoded by more than one codon. See the genetic code in Table 1. Alanine, for example, is encoded by four codons: GCU, GCC, GCA, and GCG. Three amino acids (Leu, Ser, and Arg) are encoded by six different codons, while only Trp and Met have unique codons. “Synonymous” codons are codons that encode the same amino acid. Thus, for example, CUU, CUC, CUA, CUG, UUA, and UUG are synonymous codons that code for Leu. Synonymous codons are not used with equal frequency. In general, the most frequently used codons in a particular organism are those for which the cognate tRNA is abundant, and the use of these codons enhances the rate and/or accuracy of protein translation. Conversely, tRNAs for the rarely used codons are found at relatively low levels, and the use of rare codons is thought to reduce translation rate and/or accuracy. To replace a given codon in a nucleic acid by a synonymous but less frequently used codon is to substitute a “deoptimized” codon into the nucleic acid.

Codon Bias

As used herein, a “rare” codon is one of at least two synonymous codons encoding a particular amino acid that is present in an mRNA at a significantly lower frequency than the most frequently used codon for that amino acid. Thus, the rare codon may be present at about a 2-fold lower frequency than the most frequently used codon. Preferably, the rare codon is present in at least a 3-fold, more preferably at least a 5-fold, lower frequency than the most frequently used codon for the amino acid. Conversely, a “frequent” codon is one of at least two synonymous codons encoding a particular amino acid that is present in an mRNA at a significantly higher frequency than the least frequently used codon for that amino acid. The frequent codon may be present at about a 2-fold, preferably at least a 3-fold, more preferably at least a 5-fold, higher frequency than the least frequently used codon for the amino acid. For example, human genes use the leucine codon CTG 40% of the time, but use the synonymous CTA only 7% of the time (see Table 2). Thus, CTG is a frequent codon in humans, whereas CTA is a rare codon. Roughly consistent with these frequencies of usage, there are 6 copies in the human genome for the gene for the tRNA recognizing CTG, whereas there are only 2 copies of the gene for the tRNA recognizing CTA. Similarly, human genes use the frequent codons TCT and TCC for serine 18% and 22% of the time, respectively, but the rare codon TCG only 5% of the time. TCT and TCC are read, via wobble, by the same tRNA, which has 10 copies of its gene in the human genome, while TCG is read by a tRNA with only 4 copies. It is well known that those mRNAs that are very actively translated are strongly biased to use only the most frequent codons. This includes genes for ribosomal proteins and glycolytic enzymes. On the other hand, mRNAs for relatively non-abundant proteins may use the rare codons.

The propensity for highly expressed genes to use frequent codons is called “codon bias.” A gene for a ribosomal protein might use only the 20 to 25 most frequent of the 61 codons, and have a high codon bias (a codon bias close to 1), while a poorly expressed gene might use all 61 codons, and have little or no codon bias (a codon bias close to 0). It is thought that the frequently used codons are codons where larger amounts of the cognate tRNA are expressed, and that use of these codons allows translation to proceed more rapidly, or more accurately, or both. The PV capsid protein is very actively translated, and has a high codon bias.

Codon Pair Bias

In addition to codon bias, a given organism has a preference for the nearest codon neighbor of a given codon, referred to as bias in codon pair utilization. A change in codon pair bias, without changing the existing codons, can influence the rate of protein synthesis and production of a protein.

Codon pair bias may be illustrated by considering the amino acid pair Ala-Glu, which can be encoded by 8 different codon pairs. If no factors other than the frequency of each individual codon (as shown in Table 2) are responsible for the frequency of the codon pair, the expected frequency of each of the 8 encodings can be calculated by multiplying the frequencies of the two relevant codons. For example, by this calculation in humans the codon pair GCA-GAA would be expected to occur at a frequency of 0.097 out of all Ala-Glu coding pairs (0.23×0.42; based on the frequencies in Table 2). In order to relate the expected (hypothetical) frequency of each codon pair to the actually observed frequency in the human genome the Consensus CDS (CCDS) database of consistently annotated human coding regions, containing a total of 14,795 human genes, was used. Using this set of genes, the frequencies of codon usage were re-calculated by dividing the number of occurrences of a codon by the number of all synonymous codons coding for the same amino acid. As expected the frequencies correlated closely with previously published ones such as the ones given in Table 2. Slight frequency variations are possibly due to an oversampling effect in the data provided by the codon usage database at Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon/codon.html) where 84949 human coding sequences were included in the calculation (far more than the actual number of human genes). The codon frequencies thus calculated were then used to calculate the expected codon-pair frequencies by first multiplying the frequencies of the two relevant codons with each other (see Table 3, expected frequency), and then multiplying this result with the observed frequency (in the entire CCDS data set) with which the amino acid pair encoded by the codon pair in question occurs. In the example of codon pair GCA-GAA, this second calculation gives an expected frequency of 0.098 (compared to 0.97 in the first calculation using the Kazusa dataset). Finally, the actual codon pair frequencies as observed in a set of 14,795 human genes was determined by counting the total number of occurrences of each codon pair in the set and dividing it by the number of all synonymous coding pairs in the set coding for the same amino acid pair (Table 3; observed frequency). Frequency and observed/expected values for the complete set of 3721 (61 2 ) codon pairs, based on the set of 14,795 human genes, are provided as Supplemental Table 1 in U.S. Pub. No. US2010/0209454 (Ser. No. 12/594,173) incorporated herein by reference.

If the ratio of observed frequency/expected frequency of the codon pair is greater than one, the codon pair is said to be overrepresented. If the ratio is smaller than one, it is said to be underrepresented. In Table 3, the codon pair GCA-GAA is overrepresented 1.65 fold while the coding pair GCC-GAA is more than 5-fold underrepresented.

Many other codon pairs show very strong bias in humans; some pairs are under-represented, while other pairs are over-represented. For instance, the codon pairs GCCGAA (AlaGlu) and GATCTG (AspLeu) are three- to six-fold under-represented in humans (the preferred pairs being GCAGAG and GACCTG, respectively), while the codon pairs GCCAAG (AlaLys) and AATGAA (AsnGlu) are about two-fold over-represented in humans. It is noteworthy that codon pair bias has nothing to do with the frequency of pairs of amino acids, nor with the frequency of individual codons. For instance, the under-represented pair GATCTG (AspLeu) happens to use the most frequent Leu codon, (CTG).

It has been discovered that codon pair bias diverges dramatically with increasing evolutionary distance such as between mammals and insects. For example, adjacent codon pairs under- or over-represented in humans tend to be under- or over-represented in other mammals, and adjacent codon pairs under- or over-represented in Aedes aegypti (an insect vector for certain arboviruses) tend to be under- or over-represented in certain other insects, but there is little such similarity between insects and mammals. The analysis disclosed herein revealed that codon pair preferences in insects and mammals are very different and almost uncorrelated with each other (compare FIG. 4A with FIG. 4B ). For example, in mammals the CPS of GCG GGC (Ala Gly) is +0.655, whereas in insects it is −0.651; in contrast, the CPS of CTT CCC (Leu Pro) in mammals is −0.021, whereas in insects it is +0.615. A negative codon pair score denotes that the pair is under-represented (Coleman et al., 2008), suggesting that these pairs are unfavorable for the organism. Indeed; recoding a segment of poliovirus with under-represented codon pairs yielded a dead virus (Coleman et al., 2008) even though the receded region contained the exact same synonymous codons and translated into exactly the same protein.

Codon pair observed and expected values and codon pair scores for the complete set of 3721 codon pairs in mosquito are provided in Supplemental Table 1 and are available at http://www.pnas.org/content/suppl/2015/03/24/1502864112.DCSupplemental/pnas.1502864112. sd01.pdf.

As discussed more fully below, codon pair bias takes into account the score for each codon pair in a coding sequence averaged over the entire length of the coding sequence. Codon pair bias is determined by

Accordingly, similar codon pair bias for a coding sequence can be obtained, for example, by minimized codon pair scores over a subsequence or moderately diminished codon pair scores over the full length of the coding sequence.

Calculation of Codon Pair Bias.

Every individual codon pair of the possible 3721 non-“STOP” containing codon pairs (e.g., GTT-GCT) carries an assigned “codon pair score,” or “CPS” that is specific for a given “training set” of genes in a particular organism. The CPS of a given codon pair is defined as the log ratio of the observed number of occurrences over the number that would have been expected in this set of genes (in this example the human genome). Determining the actual number of occurrences of a particular codon pair (or in other words the likelihood of a particular amino acid pair being encoded by a particular codon pair) is simply a matter of counting the actual number of occurrences of a codon pair in a particular set of coding sequences. Determining the expected number, however, requires additional calculations. The expected number is calculated so as to be independent of both amino acid frequency and codon bias similarly to Gutman and Hatfield (1989). That is, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a specific codon. A positive CPS value signifies that the given codon pair is statistically over-represented, and a negative CPS indicates the pair is statistically under-represented in the human genome.

To perform these calculations within the human context, the most recent Consensus CDS (CCDS) database of consistently annotated human coding regions, containing a total of 14,795 genes, was used. This data set provided codon and codon pair, and thus amino acid and amino-acid pair frequencies on a genomic scale.

The paradigm of Federov et al. (2002), was used to further enhanced the approach of Gutman and Hatfield (1989). This allowed calculation of the expected frequency of a given codon pair independent of codon frequency and non-random associations of neighboring codons encoding a particular amino acid pair.

In the calculation, P ij is a codon pair occurring with a frequency of N O (P ij ) in its synonymous group. C i and C j are the two codons comprising P ij , occurring with frequencies F(C i ) and F(C j ) in their synonymous groups respectively. More explicitly, F(C i ) is the frequency that corresponding amino acid X i is coded by codon C i throughout all coding regions and F(C i )=N O (C i )/N O (X i ), where N O (C i ) and N O (X i ) are the observed number of occurrences of codon C i and amino acid X i respectively. F(C j ) is calculated accordingly. Further, N O (X ij ) is the number of occurrences of amino acid pair X ij throughout all coding regions. The codon pair bias score S(P ij ) of P ij was calculated as the log-odds ratio of the observed frequency N o (P ij ) over the expected number of occurrences of N e (P ij ).

Using the formula above, it is then determined whether individual codon pairs in individual coding sequences are over- or under-represented when compared to the corresponding genomic N e (P ij ) values that were calculated by using the entire human CCDS data set (or data set from another host). This calculation provides positive S(P ij ) score values for over-represented and negative values for under-represented codon pairs in the human coding regions.

The “combined” codon pair bias of an individual coding sequence is calculated by averaging all codon pair scores according to the following formula:

The codon pair bias of an entire coding region is thus calculated by adding all of the individual codon pair scores comprising the region and dividing this sum by the length of the coding sequence.

Calculation of Codon Pair Bias, Implementation of Algorithm to Produce Codon Pair Deoptimized Sequences (Relative to a Single Host).

An algorithm was developed to quantify codon pair bias. Every possible individual codon pair was given a “codon pair score,” or “CPS”. CPS is defined as the natural log of the ratio of the observed over the expected number of occurrences of each codon pair over all coding regions in a particular organism.

Although the calculation of the observed occurrences of a particular codon pair is straightforward (the actual count within the gene set), the expected number of occurrences of a codon pair requires additional calculation. This expected number is calculated to be independent both of amino acid frequency and of codon bias, similar to Gutman and Hatfield. That is, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a specific codon. A positive CPS value signifies that the given codon pair is statistically over-represented, and a negative CPS indicates the pair is statistically under-represented in the human genome.

Using these calculated CPSs, any coding region can then be rated as using over- or under-represented codon pairs by taking the average of the codon pair scores, thus giving a Codon Pair Bias (CPB) for the entire coding sequence.

Accordingly, similar codon pair bias for a coding sequence can be obtained, for example, by minimized codon pair scores over a subsequence or moderately diminished codon pair scores over the full length of the coding sequence.

Codon pair bias, which is a measure of codon pair usage, can be evaluated for a coding sequence, whether or not codon substitutions are made as described below. In addition to the above example of CPB relative to humans, CPB relative to other organisms can be calculated utilizing the reference genome sequence for that organism.

Virus Attenuation by Optimization/Deoptimization Codon Pair Bias Relative to Two Hosts

The present invention utilizes the differences in codon pair bias between two viral hosts that are phlogenetically distinct to recode one or more virus protein-encoding sequences by manipulating the codon pair bias of the sequence(s) relative to the two hosts. The one or more virus protein-encoding sequences are recoded to (i) deoptimize the virus for growth in one host while optimizing (or leaving unchanged) growth in another host; (ii) deoptimize the virus for growth in both hosts; or (iii) optimize the virus for growth in both hosts. The methods of the present invention are used to generate one or more modified virus protein-encoding sequences wherein the codon pair bias, relative to a first host, is less than the codon pair bias of the parent nucleic acid from which it is derived (e.g., the wild-type sequence), and wherein the codon pair bias of the one or more modified virus protein-encoding sequences is not substantially reduced relative to that of a second host. The methods of the present invention are also used to generate one or more modified virus protein-encoding sequences wherein the codon pair bias, relative to both hosts, is less than the codon pair bias of the parent nucleic acid from which it is derived (e.g., the wild-type sequence).

In one embodiment, the virus comprises a protein encoding nucleic acid sequence that is recoded to deoptimize the sequence with respect to replication in a mammalian host or cell, while optimizing its replication properties in an insect host or cell. In another embodiment, a protein encoding nucleic acid sequence is recoded to deoptimize the sequence with respect to expression in a mammalian host of cell, and its replication properties in an insect host or cell are also deoptimized. Similarly, in other embodiments, protein encoding nucleic acid sequences can be recoded to maintain or optimize replication in a mammalian host or cell while deoptimizing, maintaining, or optimizing replication properties in an insect host or cell.

According to the invention, viruses can be described, by their replication properties. Maintaining replicative properties in a particular host means viral titers obtained for the recoded virus at levels similar to, e.g., within 2× or 5× of viral titers of the parent virus. Deoptimizing a virus with respect to a host means reducing viral titers by 5× or more, for example 10×, 50×, 100×, 500×, or 1000× or more. Likewise, optimizing a virus with respect to a host means increasing viral titers by 5× or more, for example 10×, 50×, 100×, 500×, or 1000× or more.

According to the invention, codon pair bias can be altered independently of codon usage. For example, in a protein encoding sequence of interest, codon pair bias can be altered simply by directed rearrangement of its codons. In particular, the same codons that appear in the parent sequence, which can be of varying frequency in the host organisms, are used in the altered sequence, but in different positions. In the simplest form, because the same codons are used as in the parent sequence, codon usage over the protein coding region being considered remains unchanged (as does the encoded amino acid sequence). Nevertheless, certain codons appear in new contexts, that is, preceded by and/or followed by codons that encode the same amino acid as in the parent sequence, but employing a different nucleotide triplet.

The rearrangement of a codon may result in two codon pairs that are both less frequent in a host than in the parent sequence. In practice, rearranging codons often results in a less frequent codon pair at one location and a more frequent pair at a second location. By judicious rearrangement of codons, the codon pair usage bias over a given length of coding sequence can be reduced relative to the parent sequence. Alternatively, the codons could be rearranged so as to produce a sequence that makes use of codon pairs which are more frequent in the host than in the parent sequence.

Codon pair bias is evaluated by considering each codon pair in turn, scoring each pair according to the frequency that the codon pair is observed in protein coding sequences of a host, and then determining the codon pair bias for the sequence, as disclosed herein. It will be appreciated that one can create many different sequences that have the same codon pair bias. Also, codon pair bias can be altered to a greater or lesser extent, depending on the way in which codons are rearranged. The codon pair bias of a coding sequence can be altered by recoding the entire coding sequence, or by recoding one or more subsequences. As used herein, “codon pair bias” is evaluated over the length of a coding sequence, even though only a portion of the sequence may be mutated. Because codon pairs are scored in the context of codon usage of the host organism, a codon pair bias value can be assigned to wild type viral sequences and mutant viral sequences. A virus can be attenuated by recoding all or portions of the protein encoding sequences of the virus so as to reduce its codon pair bias.

Codon pair bias is a quantitative property determined from codon pair usage of a host. Accordingly, absolute codon pair bias values may be determined for any given viral protein coding sequence for a given host. And a viral protein encoding sequence may have different absolute codon pair bias values relative to different hosts, in particular when the different hosts are phylogenetically distinct (for example the hosts are from different kingdoms phyla, or classes). Alternatively, relative changes in codon pair bias values can be determined that relate a deoptimized viral protein coding sequence to a “parent” sequence from which it is derived. As viruses come in a variety of types (i.e., types I to VII by the Baltimore classification), and natural (i.e., virulent) isolates of different viruses yield different values of absolute codon pair bias, it is relative changes in codon pair bias that are usually more relevant to determining desired levels of attenuation for a given host. Accordingly, the invention provides attenuated viruses and methods of making such, wherein the attenuated viruses comprise viral genomes in which one or more protein encoding nucleotide sequences have codon pair bias reduced by mutation. In viruses that encode only a single protein (i.e., a polyprotein), all or part of the polyprotein can be mutated to a desired degree to reduce codon pair bias, and all or a portion of the mutated sequence can be provided in a recombinant viral construct. For a virus that separately encodes multiple proteins, one can reduce the codon pair bias of all of the protein encoding sequences simultaneously, or select only one or a few of the protein encoding sequences for modification. The reduction in codon pair bias is determined over the length of a protein encoding sequences, and is at least about 0.05, or at least about 0.1, or at least about 0.15, or at least about 0.2, or at least about 0.3, or at least about 0.4. Depending on the virus, the absolute codon pair bias, based on codon pair usage of the host, can be about −0.05 or less, or about 0.1 or less, or about −0.15 or less, or about −0.2 or less, or about −0.3 or less, or about −0.4 or less.

Viruses of the invention can be described by shifts in codon-pair preference or codon pair bias. As describe herein, codon-pair score (CPS) and codon-pair bias (CPB) relate to the observed vs. expected frequency of adjacent codon pairs in a host. For viruses of the invention, which replicate in more than one host, CPS and CPB are evaluated independently for each host. Virus protein encoding nucleic acid sequences that are deoptimized for a particular host can have reduced CPB, in other words CPB values that are substantially more negative than the parent virus protein encoding sequence evaluated for that host. For example, the attenuated virus can have CPB scores that are at least, 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, from 0.5 to 0.1, from 0.1 to 0.2, from 0.2 to 0.3, from 0.3 to 0.4, or from 0.5 to 0.5 more negative than parent viruses evaluated for that host. According to the invention, a nucleic acid sequence may be deoptimized and have a CPB that is reduced for one host, without a substantial change in CPB for a second host. For example, the CPB with respect to the second host can be within 0.002, 0.005, 0.010, 0.020, or 0.050 of the parent virus with respect to the second host. The above values are not strict limitations on changes in CPB values, as effects on replication may vary depending on which coding sequences of the virus are modified. While in certain embodiments, changes in CPB compared to a parent virus result from rearrangement, or shuffling, of the parent virus codons, in other embodiments, the recoded virus can also contain synonymous codon substitutions and/or encode amino acid substitutions.

It will be apparent that codon pair bias can also be superimposed on other sequence variation. For example, a coding sequence can be altered to encode a protein or polypeptide which contains one or more amino acid changes and also has been recoded by shuffling synonymous codons in order to alter codon pair bias. Also, one may shuffle codons to maintain exactly the same codon usage profile in a codon pair bias reduced protein encoding sequence as in a parent protein encoding sequence. Alternatively, codon selection can result in an overall change in codon usage in a coding sequence.

According to the invention, viral attenuation can be accomplished by changes in codon pair bias as well as codon bias. Both deoptimized codon bias, and deoptimized codon pair bias, separately create non-viable viruses probably by causing inefficient translation of the recoded sequence. However, it is expected that adjusting codon pair bias is particularly advantageous. For example, attenuating a virus through codon bias generally requires elimination of common codons, and so the complexity of the nucleotide sequence is reduced. In contrast, codon pair bias reduction or minimization can be accomplished while maintaining far greater sequence diversity, and consequently greater control over nucleic acid secondary structure, annealing temperature, and other physical and biochemical properties. The work disclosed herein includes attenuated codon pair bias-reduced or -minimized sequences in which codons are shuffled, but the codon usage profile is unchanged or substantially unchanged.

During recoding, essential nucleic acid signals in the viral genome are preserved, but the efficiency of protein translation in one or both hosts is systematically reduced by deoptimizing codon pair bias. Other parameters may also be deoptimized such as codon bias, RNA secondary structure and CpG dinucleotide content, C+G content, translation frameshift sites, translation pause sites, or any combination thereof. This deoptimization may involve hundreds or thousands of changes, each with a small effect. Generally, deoptimization is performed to a point at which the virus can still be grown in some cell lines (including lines specifically engineered to be permissive for a particular virus), but where the virus is avirulent in one or more host. Such avirulent viruses are excellent candidates for either a killed or live vaccine since they encode exactly the same proteins as the fully virulent virus and accordingly provoke exactly the same immune response as the fully virulent virus. In addition, the present invention offers the prospect for fine tuning the level of attenuation in each host; that is, it provides the capacity to design synthetic viruses that are deoptimized to a roughly predictable extent in one or more hosts. Design, synthesis, and production of viral particles is achievable in a timeframe of weeks once the genome sequence is known, which has important advantages for the production of vaccines in potential emergencies. Furthermore, the attenuated viruses are expected to have virtually no potential to revert to virulence because of the extremely large numbers of deleterious nucleotide changes involved.

The extent and intensity of recoding of a virus can be varied depending on the length of the protein encoding nucleic acid, whether all or a portion can be recoded, and the desired reduction of codon pair bias. In an embodiment of the invention, a protein encoding sequence is modified over a length of at least about 100 nucleotides, or at least about 200 nucleotides, or at least about 300 nucleotides, or at least about 500 nucleotides, or at least about 1000 nucleotides.

A attenuated virus according to the present invention has been modified to reduce pathogenicity in one or more hosts. The attenuated virus has reduced virulence in one or more hosts, but can stimulate an immune response in a subject. Viral attenuation can be confirmed in ways that are well known to one of ordinary skill in the art. Non-limiting examples include plaque assays, growth measurements, and reduced lethality in test animals. The instant application demonstrates that the attenuated viruses are capable of inducing protective immune responses in a host.

The term “parent” virus or “parent” protein encoding sequence is used herein to refer to viral genomes and protein encoding sequences from which new sequences, which may be more or less attenuated, are derived. Parent viruses and sequences are usually “wild type” or “naturally occurring” prototypes or isolates of variants for which it is desired to obtain a more highly attenuated virus. However, parent viruses also include mutants specifically created or selected in the laboratory on the basis of real or perceived desirable properties. Accordingly, parent viruses that are candidates for attenuation include mutants of wild type or naturally occurring viruses that have deletions, insertions, amino acid substitutions and the like, and also include mutants which have codon substitutions. In one embodiment, such a parent sequence differs from a natural isolate by about 30 amino acids or fewer. In another embodiment, the parent sequence differs from a natural isolate by about 20 amino acids or fewer. In yet another embodiment, the parent sequence differs from a natural isolate by about 10 amino acids or fewer.

The description of a virus as having a first host and a second host is not meant to imply order of infection or any relative value of the two hosts. Rather, the use of the terms first host and second host identifies viral hosts that are phylogenetically distant and thus have sufficiently different codon pair preferences that the viral sequence can be manipulated to, for example, simultaneously favor one host over the other. In one embodiment the first host and second host are from different kingdoms. In another embodiment, the first host and second host are from different phyla. In another embodiment first host and second host are from different classes.

Algorithm to Produce Recoded Sequences with CPB Rebalanced Relative to Two Hosts.

As exemplified herein, a computer-based algorithm can be used to manipulate the codon pair bias of any coding region relative to two hosts. The algorithm has the ability to shuffle existing codons and to evaluate the resulting CPB relative to two hosts, and then to reshuffle the sequence, optionally locking in particularly “valuable” codon pairs. The algorithm also employs a form of “simulated annealing” so as not to get stuck in local minima. Other parameters, such as the free energy of folding of RNA, may optionally be under the control of the algorithm as well, in order to avoid creation of undesired secondary structures. The algorithm can be used to find a sequence with codon pair bias that is independently minimized, maximized, or substantially unchanged, relative to two unrelated hosts. In the event that such a sequence does not provide a viable virus, the algorithm can be adjusted to find sequences with reduced, but not minimized biases.

Choosing a random codon and swapping it with another randomly chosen synonymous codon, the heuristic works over a particular sequence in several hundred thousand iterations. If the codon change is “good”, the change is retained, while if the change is “bad,” it may still be retained, with a probability dependent on a specified “temperature” (hence the analogy to metallurgical annealing). Unlike the codon pair deoptimization for a single host described previously, in this case there is a non-trivial bi-criteria optimization problem, in which, for example, the cumulative score of codon pairs is minimized according to the human codon pair bias table, while not allowing the cumulative score according to the insect table to drift substantially. Combining both criteria into a single function:

min( a *human_score+ b *abs(insect_score−insect_score_wt) c )

where a, b, and c are coefficients. By varying a, b, and c, one can, for example, control the importance of minimizing the human score (a), and limiting the variation of the insect score from wild-type (b and c). The same approach may be used to simultaneously reduce the codon pair score relative to both hosts to produce a virus that is attenuated in, for example, both humans and insects.

Sequence optimization/deoptimization relative to two hosts may be performed with or without the aid of a computer, using, for example, a gradient descent, or simulated annealing, or other minimization routine. An example of the procedure that rearranges codons present in a starting sequence can be represented by the following steps:

(1) Obtain parent (e.g., wild-type) viral genome sequence.

(2) Select protein encoding sequence(s) to target for attenuated design.

(3) Lock down known or conjectured DNA segments with non-coding functions.

(4) Select heuristic function coefficients to determine relative importance of minimizing CPB score relative to a first host versus keeping neutral CPB score relative to a second host; or alternatively minimizing CPB scores relative to a first host and relative to a second host.

(5) Perform random shuffle of at least two synonymous unlocked codon positions and calculate two codon-pair bias scores relative to a first and second host.

(6) Compute the resulting change in heuristic function (e.g., by simulated annealing) and keep or reject the synonymous codon shuffle.

(7) Repeat steps (5) and (6) for desired number of iterations.

In addition to the above steps, one or more of the following steps may be performed to generate a virus that has altered growth properties relative to at least one of two hosts:

(8) Inspect resulting design for excessive secondary structure and unwanted restriction site:

if yes->go to step (5) or correct the design by replacing problematic regions with wildtype sequences and go to step (9).

(9) Synthesize DNA sequence corresponding to virus design.

(10) Create viral construct and assess viral phenotype:

if too attenuated, prepare subclone construct and go to 10; if insufficiently attenuated, go to 2.

Using the formulas above, a computer based algorithm was developed to manipulate the CPB of any coding region with respect to two hosts having divergent codon pair preferences while maintaining the original amino acid sequence. The algorithm has the ability to maintain the codon usage of the selected sequence (i.e. preserve the frequency of use of each existing codon) but “shuffle” the existing codons so that the CPB can be increased, decreased, or remain substantially unchanged relative to each of the two hosts. The algorithm uses simulated annealing, a mathematical process suitable for full-length optimization (Park, et al., 2004). Other parameters are also under the control of this algorithm; for instance, the free energy of the folding of the RNA. This free energy is maintained within a narrow range, to prevent large changes in secondary structure as a consequence of codon re-arrangement. The optimization process specifically excludes the creation of any regions with large secondary structures, such as hairpins or stem loops, which could otherwise arise in the customized RNA. Using this computer software the user simply needs to input the cDNA sequence of a given gene and the CPB of the gene can be customized as the experimenter sees fit.

Alternatively, one can devise a procedure which allows each pair of amino acids to be deoptimized by choosing a codon pair without a requirement that the codons be swapped out from elsewhere in the protein encoding sequence.

This invention provides a method of making an attenuated virus genome, the method comprising: (a) obtaining an virus protein-encoding sequence; (b) rearranging synonymous codons of the nucleotide sequences to obtain modified nucleotide sequences that (i) encodes the same amino acid sequence as the unrearranged nucleotide sequence, (ii) has a reduced codon pair bias relative to a first host compared to the unrearranged nucleotide sequence, (iii) has a substantially similar codon pair bias or a reduced codon pair bias relative to a second host compared to the unrearranged nucleotide sequence; and (c) substituting all or part of the modified nucleotide sequence into the unrearranged genome of a parent virus.

In certain embodiments of the instant methods, step (b) is guided by a computer-based algorithm described above that permits design of a viral genome by varying specified pattern sets of deoptimized codon distribution and/or deoptimized codon-pair distribution within preferred limits. The invention also provides a method wherein, the pattern sets alternatively or additionally comprise, density of deoptimized codons and deoptimized codon pairs, RNA secondary structure, CpG dinucleotide content, C+G content, overlapping coding frames, restriction site distribution, frameshift sites, or any combination thereof.

In one embodiments, the recoded viral protein encoding sequence is generated by de novo synthesis of DNA containing the synonymous codons and/or codon pairs.

Attenuated Arboviruses

Arboviruses, for example, exhibit life cycles that involve both vertebrates and arthropods as hosts. In order to infect and replicate in these two very different types of hosts, the virus must be able to adapt to growth conditions that are very different, including temperature, host factors, cell membrane thickness and composition, and even differences in genome synonymous codon usage and codon pair bias.

One aspect of the present invention involves “recoding” of arbovirus genomes including but not limited to DENV so as to alter or disrupt the finely balanced use of codon pairs that permits the virus to efficiently use both insect and mammalian translational machineries. In one embodiment, codon pairings are utilized that are similarly favorable as the wild-type virus for expression in insects (thus allowing for vaccine production in insect cell culture) while, at the same time, being detrimental for expression in human host (attenuation). In another embodiment, the disclosed technique can be used to produce arboviruses that are attenuated in both host as vaccine candidates.

In one embodiment, the attenuated virus of the present invention is an arbovirus. Families in the current classification system that have some arbovirus members include Bunyaviridae (comprising the bunyaviruses, phleboviruses, nairoviruses, and hantaviruses), Flaviviridae (comprising only the flaviviruses), Reoviridae (comprising the coltiviruses and orbiviruses), and Togaviridae (comprising the alphaviruses). Birds are often reservoirs for arboviruses, which are transmitted by mosquitoes to horses, other domestic animals, and humans. Certain arboviruses are transmissible by humans, including dengue fever, yellow fever, and chikungunya disease, which can be transmitted from person to person via mosquitoes.

In one embodiment of the invention the arbovirus is yellow fever virus, West Nile virus, dengue virus, chikungunya virus, African swine fever virus, Japanese encephalitis virus, Rift Valley fever virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, Bunyamwera virus, California encephalitis virus, Jamestown Canyon virus, La Crosse encephalitis, Toscana virus, heartland virus, Kyasanur forest disease virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, African horse sickness virus, bluetongue disease virus, equine encephalosis virus, banna virus, Coltivirus Colorado tick fever virus, Eastern equine encephalitis virus, Ross River virus, Venezuelan equine encephalitis virus, and Western equine encephalitis virus.

In one embodiment the arbovirus is dengue virus. There are four dengue virus serotypes (DENV 1-4) that commonly infect humans. A fifth DENV serotype has recently been reported in Malaysia, although only one human infection has been documented.

According to the invention, one or more virus protein-encoding nucleic acid sequences, or portions thereof, may be modified. In this regard, for example, dengue virus encodes several proteins in an open reading frame: C; prM; E; NS1; 2A; 2B; NS3; 4A; 4B, and NS5. DENV C is a capsid protein; the DENV E (envelope) protein is found on the viral surface and is important in the initial attachment of the viral particle to the host cell; the DENV prM (membrane) protein is important in the formation and maturation of the viral particle; DENV NS3 is a serine protease, as well as an RNA helicase and RTPase/NTPase; DENV NS5 is a 900 residue peptide with a methyltransferase domain at its N-terminal end and a RNA-dependent RNA polymerase (RdRp) at its C-terminal end; NS4B it is a small hydrophobic protein which may block phosphorylation of STAT1 and inhibit interferon signaling; NS5 inactivates and degrades STAT2.

The invention is exemplified by recoding of the E structural glycoprotein (SEQ ID NO:3, recoded E), NS3 multi-functional protease (SEQ ID NO:4, recoded NS3), and NS5 multifunctional RNA polymerase (SEQ ID NO:5, recoded NS5) of the DENV serotype 2 (strain 16681; SEQ ID NO:1). As exemplified, a DENV serotype 2 virus genome based on the 16681 strain was synthesized ab initio with 26 silent nucleotide changes to provide convenient restriction sites (SEQ ID NO:2) (referred to herein as “D2-syn” and also called D2SAM1). The growth characteristics of the synthesized virus in monkey and mosquito cells are indistinguishable from the 16681 strain. The virus genome was recoded in silico to produce three recoded open reading frames having strongly negative human codon pair scores compared to wild-type, but mosquito codon pair scores similar to wild-type.

Accordingly, the invention provides arboviruses adapted for use in vaccines, as well as methods of making and using such viruses. According to the invention, protein encoding sequences of the virus can be recoded to alter replication properties in one or more of its mammalian and insect hosts. In one embodiment, the virus comprises a protein encoding nucleic acid sequence that is recoded to deoptimize the sequence with respect to replication in a mammalian host or cell, while maintaining its replication properties in an insect host or cell. In one embodiment, the invention provides an arbovirus that is attenuated in a human, and can be produced at high titers in cells of a second, evolutionarily distant, host. For example, existing synonymous codons of an arbovirus are rearranged so as to replace existing adjacent codon pairs with pairs that are unfavorable in humans and favorable in insects.

Large-Scale DNA Assembly

In recent years, the plunging costs and increasing quality of oligonucleotide synthesis have made it practical to assemble large segments of DNA (at least up to about 10 kb) from synthetic oligonucleotides. Commercial vendors such as Blue Heron Biotechnology, Inc. (Bothwell, Wash.) (and also many others) currently synthesize, assemble, clone, sequence-verify, and deliver a large segment of synthetic DNA of known sequence for the relatively low price of about $1.50 per base. Thus, purchase of synthesized viral genomes from commercial suppliers is a convenient and cost-effective option, and prices continue to decrease rapidly. Furthermore, new methods of synthesizing and assembling very large DNA molecules at extremely low costs are emerging (Tian et al., 2004). The Church lab has pioneered a method that uses parallel synthesis of thousands of oligonucleotides (for instance, on photo-programmable microfluidics chips, or on microarrays available from Nimblegen Systems, Inc., Madison, Wis., or Agilent Technologies, Inc., Santa Clara, Calif.), followed by error reduction and assembly by overlap PCR. These methods have the potential to reduce the cost of synthetic large DNAs to less than 1 cent per base. The improved efficiency and accuracy, and rapidly declining cost, of large-scale DNA synthesis provides an impetus for the development and broad application of the creation of attenuated virus by the strategies disclosed herein.

Vaccine Compositions

The present invention provides a vaccine composition for inducing the production of neutralizing antibodies in a subject. In one embodiment, the present invention provides a vaccine composition for inducing a protective immune response in a subject comprising any of the attenuated viruses described herein and a pharmaceutically acceptable carrier. In one aspect of the invention the attenuated virus is an arbovirus. In a further embodiment the attenuated virus is a dengue virus. In one aspect of the invention, vaccine composition comprises an attenuated DENV and is affective at inducing protective immunity against one or more DENV serotypes. In one aspect, the vaccine composition comprises one or more DENV serotypes. In one embodiment, the attenuated arbovirus is a chimeric construct (see Caufour et al, 2001; Osorio et al., 2011; Durbin et al., 2011) used to develop a multivalent (e.g., tetravalent) vaccine.

In an embodiment of the invention, a vaccine composition is provided for inducing a protective immune response in a subject, wherein the vaccine composition comprises an arbovirus as set forth above. In an embodiment of the invention, the vaccine composition further comprises at least one adjuvant. The invention provides a method of eliciting a protective immune response in a subject comprising administering to the subject a prophylactically or therapeutically effective dose of a vaccine composition set forth above.

It should be understood that an attenuated virus of the invention, where used to elicit a protective immune response in a subject or to prevent a subject from becoming afflicted with a virus-associated disease, is administered to the subject in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well 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), 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 and 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 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 of the instant vaccine composition, the attenuated virus (i) does not substantially alter the synthesis and processing of viral proteins in an infected cell; (ii) produces similar amounts of virions per infected cell as wild-type virus; and/or (iii) exhibits substantially lower virion-specific infectivity than wild-type virus. In further embodiments, the attenuated virus induces a substantially similar immune response in a host animal as the corresponding wt virus.

This invention also provides a modified host cell line specially isolated or engineered to be permissive for an attenuated virus that is inviable in a wild type host cell or otherwise not efficiently replicated in cell culture. Since the attenuated virus cannot grow in normal (wild type) host cells, it is dependent on the specific helper cell line for growth. This provides a very high level of safety for the generation of virus for vaccine production. Various embodiments of the instant modified cell line permit the growth of an attenuated virus, wherein the genome of said cell line has been altered to increase the number of genes encoding rare tRNAs.

In addition, the present invention provides a method for eliciting a protective immune response in a subject comprising administering to the subject a prophylactically or therapeutically effective dose of any of the vaccine compositions described herein. This invention also provides a method for preventing a subject from becoming afflicted with a virus-associated disease comprising administering to the subject a prophylactically effective dose of any of the instant vaccine compositions. In embodiments of the above methods, the subject has been exposed to a pathogenic virus. “Exposed” to a pathogenic virus means contact with the virus such that infection could result.

The invention further provides a method for delaying the onset, or slowing the rate of progression, of a virus-associated disease in a virus-infected subject comprising administering to the subject a therapeutically effective dose of any of the instant vaccine compositions.

As used herein, “administering” means delivering using any of the various methods and delivery systems known to those skilled in the art. Administering can be performed, for example, intraperitoneally, intracerebrally, intravenously, orally, transmucosally, subcutaneously, transdermally, intradermally, intramuscularly, topically, parenterally, via implant, intrathecally, intralymphatically, intralesionally, pericardially, or epidurally. An agent or composition may also be administered in an aerosol, such as for pulmonary and/or intranasal delivery. Administering may be performed, for example, once, a plurality of times, and/or over one or more extended periods.

Eliciting a protective immune response in a subject can be accomplished, for example, by administering a primary dose of a vaccine to a subject, followed after a suitable period of time by one or more subsequent administrations of the vaccine. A suitable period of time between administrations of the vaccine may readily be determined by one skilled in the art, and is usually on the order of several weeks to months. The present invention is not limited, however, to any particular method, route or frequency of administration.

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. In a preferred embodiment, the subject is a human.

A “prophylactically effective dose” is any amount of a vaccine that, when administered to a subject prone to viral infection or prone to affliction with a virus-associated disorder, induces in the subject an immune response that protects the subject from becoming infected by the virus or afflicted with the disorder. “Protecting” the subject means either reducing the likelihood of the subject's becoming infected with the virus, or lessening the likelihood of the disorder's onset in the subject, by at least two-fold, preferably at least ten-fold. For example, if a subject has a 1% chance of becoming infected with a virus, a two-fold reduction in the likelihood of the subject becoming infected with the virus would result in the subject having a 0.5% chance of becoming infected with the virus. Most preferably, a “prophylactically effective dose” induces in the subject an immune response that completely prevents the subject from becoming infected by the virus or prevents the onset of the disorder in the subject entirely.

As used herein, a “therapeutically effective dose” is any amount of a vaccine that, when administered to a subject afflicted with a disorder against which the vaccine is effective, induces in the subject an immune response that causes the subject to experience a reduction, remission or regression of the disorder and/or its symptoms. In preferred embodiments, recurrence of the disorder and/or its symptoms is prevented. In other preferred embodiments, the subject is cured of the disorder and/or its symptoms.

Certain embodiments of any of the instant immunization and therapeutic methods further comprise administering to the subject at least one adjuvant. An “adjuvant” shall mean any agent suitable for enhancing the immunogenicity of an antigen and boosting an immune response in a subject. Numerous adjuvants, including particulate adjuvants, suitable for use with both protein- and nucleic acid-based vaccines, and methods of combining adjuvants with antigens, are well known to those skilled in the art. Suitable adjuvants for nucleic acid based vaccines include, but are not limited to, Quil A, imiquimod, resiquimod, and interleukin-12 delivered in purified protein or nucleic acid form. Adjuvants suitable for use with protein immunization include, but are not limited to, alum, Freund's incomplete adjuvant (FIA), saponin, Quil A, and QS-21.

The invention also provides a kit for immunization of a subject with an attenuated virus of the invention. The kit comprises the attenuated virus, a pharmaceutically acceptable carrier, an applicator, and an instructional material for the use thereof. In further embodiments, the attenuated virus may be one or more poliovirus, one or more rhinovirus, one or more influenza virus, etc. More than one virus may be preferred where it is desirable to immunize a host against a number of different isolates of a particular virus. The invention includes other embodiments of kits that are known to those skilled in the art. The instructions can provide any information that is useful for directing the administration of the attenuated viruses.

Throughout this application, various publications, reference texts, textbooks, technical manuals, patents, and patent applications have been referred to. The teachings and disclosures of these publications, patents, patent applications and other documents in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which the present invention pertains. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention.

It is to be understood and expected that variations in the principles of invention herein disclosed can be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention. The following Examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed in the construction of recombinant plasmids, transfection of host cells with viral constructs, polymerase chain reaction (PCR), and immunological techniques can be obtained from numerous publications, including Sambrook et al. (1989) and Coligan et al. (1994). All references mentioned herein are incorporated in their entirety by reference into this application.

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