VIRUS ATTENUATION

Abstract

The present disclosure relates to paramyxoviruses, in particular attenuated avian avulaviruses (para, ortho and meta), mutated and genetically modified forms, as well as a vaccine formulation comprising an attenuated avian avulavirus and uses/methods of use thereof.

Claims

1. An attenuated velogenic avian orthoavulavirus (AOaV), wherein each of the HN and F genes of an AOaV genome of the attenuated AOaV comprises, consists essentially of, or consists of a plurality of silent mutations, as compared to a wild-type or parent AOaV from which the attenuated AOaV has been derived.

2. The attenuated AOaV according to claim 1, wherein the silent mutations have been obtained by codon deoptimsation strategies.

3. The attenuated AOaV according to claim 2, wherein the codon optimisation strategy comprises using a Smart Codon Usage Algorithm (SCUA): $\mathrm{SCUA}=\frac{\frac{\mathrm{CfOt}}{\mathrm{CfOr}}\mathrm{NOt}}{\mathrm{NOr}}\mathrm{vgf}$ Here, CfOt: frequency of codon occurrence in test sequence CfOr: frequency of codon occurrence in reference sequence NOt: number of codon occurrences in test sequence NOr: number of codon occurrences in reference sequence vgf: viral genomic features.

4. The attenuated AOaV according to either of claim 1 or 2 wherein each of the HN and F genes includes at least 5, 10, 15, 20, 25, 30, 40, 50 or more mutated codons.

5. The attenuated AOaV according to any preceding claim comprising one or more substitutions, inversions, deletions, or additions in any one or more of the NP, P, M, or L genes.

6. The attenuated AOaV according to any preceding claim, which has been further modified in order to express one or more proteins or antigenic fragments thereof, from another pathogen, such as another virus, or organism.

7. A vaccine or pharmaceutical composition comprising the attenuated AOaV according to any preceding claim, together with a pharmaceutically acceptable excipient therefor.

8. A modified AOaV genome encoding for an attenuated AOaV according to any of claims 1-5.

9. A vector comprising the modified AOaV genome according to claim 7.

10. The attenuated AOaV, vaccine or pharmaceutical composition, modified AOaV genome or vector according to any preceding claim, for use in therapy.

Description

DETAILED DESCRIPTION OF THE DISCLOSURE

[0074] The present disclosure will now be further described by way of non-limiting example and with reference to the figures, which show:

[0075] FIG. 1. A-Schematic representation of wild type (wt) and codon optimized (CD) AOaV-1. (A) rAOaV-1-WT contains genes in the order from N, P, M, F, HN and L. The rAOaV-1-F.sub.CD contain full-length open reading frame of F which is codon deoptimized while rest of all genes carry sequence of rAOaV-1-WT. Similarly, the codon deoptimized HN gene replaced the corresponding WT in the construct rAOaV-1-HN.sub.CD. The rAOaV-1-F.sub.CD+HN.sub.CD contains both HN and F gene codon deoptimized while rest of all genes remained unmodified except that the F protein was modified at the cleavage site; Bplaque assays showing the replication capacity of wild-type and mutated rAOaV viruses described herein; and Cgraph showing the quantitative measurement of the plaques in FIG. 1B.

[0076] FIG. 2. Reduced expression levels of F gene in transfected cells. (A) Immunofluorescence of F.sub.WT gene in transfected cells and immunofluorescence of F.sub.CD gene in transfected cells in increasing concentrations. (B) Western blot of F.sub.WT and F.sub.CD in transfected cells with increasing concentration. Alpha tubulin was used as loading control.

[0077] FIG. 3. Western blot expression of HN.sub.WT and HN.sub.CD in transfected cells with increasing concentration of 250 ng, 500 ng and 750 ng. Alpha tubulin was used as loading control.

[0078] FIG. 4. In vitro characterization of rAOaV-1. (A) Immunofluorescence of rAOaV-1-F.sub.CD, rAOaV-1-HN.sub.CD and rAOaV-1-F.sub.CD+HN.sub.CD with antibodies against F protein.

[0079] FIG. 5. In ovo attenuation of rAOaV-1 in chicken eggs. (A) The replication of rAOaV-1-F.sub.CD+HN.sub.CD replication after first and tenth passage in embryonated chicken eggs. (B and C) The replication kinetics by RT-PCR show a comparable replication of rAOaV-1-F.sub.WT, rAOaV-1-F.sub.CD, rAOaV-1-HN.sub.CD and rAOaV-1-F.sub.CD+HN.sub.CD at first (B) and tenth (C) passage. (D and E) All recombinant viruses (rAOaV-1-F.sub.WT, rAOaV-1-F.sub.CD, rAOaV-1-HN.sub.CD and rAOaV-1-F.sub.CD+HN.sub.CD showed similar replication profile by plaque assays (PFU/ml) at first (D) and tenth (E) passage.

[0080] FIG. 6. Attenuation of rAOaV-1 in chicken. (A ans B) A low weight loss was noticed in chicken infected with rAOaV-1-F.sub.CD+HN.sub.CD compared to individual or no codon deoptimized genes containing recombinant viruses (rAOaV-1-F.sub.CD, and rAOaV-1-HN.sub.CD) at both high doses (A) and low doses (B). (C) The rAOaV-1-F.sub.CD+HN.sub.CD showed reduced mortality and appeared safe compared to rAOaV-1-F.sub.WT, rAOaV-1-F.sub.CD, rAOaV-1-HN.sub.CD. (D) A similar trend was observed when a high dose was used. (E) Replication analysis of rAOaV-1-F.sub.WT, rAOaV-1-F.sub.CD, rAOaV-1-HN.sub.CD and rAOaV-1-F.sub.CD+HN.sub.CD in chicken showed attenuated replication of rAOaV-1-F.sub.CD+HN.sub.CD. (F) A corresponding replication of rAOaV-1-F.sub.CD+HN.sub.CD was observed when higher level of infection was used.

[0081] FIG. 7. Protection efficacy of recombinant rAOaV-1. (A) Experimental plan to demonstrate the protective efficacy of recombinant viruses. (B) HA titre in rAOaV-1-F.sub.CD+HN.sub.CD vaccinated compared to non-vaccinated animals. (C) The rAOaV-1-F.sub.CD+HN.sub.CD was attenuated and showed full protection against pathogenic viruses compared. (D) Correspondingly, a low weight loss was observed in animals infected with rAOaV-1-F.sub.CD+HN.sub.CD. (E) The HA titre before culling of animals indicate high antibodies in rAOaV-1-F.sub.CD+HN.sub.CD vaccinated animals compared to non-vaccinated animals.

[0082] FIG. 8. A lower level of pathological changes was noticed in chicken trachea and lung infected with rAOaV-1-F.sub.CD+HN.sub.CD vaccinated compared to mock-vaccinated and challenged, and mock-vaccinated and mock challenged animals.

EXAMPLE 1

[0083] In order to design and produce the codon deoptimized rAOaV-1, the F and HN genes were codon deoptimized through the SCUA algorithm described herein. The original rAOaV-1-WT nucleotide sequences were maintained all other open reading frame, other than HN and F. Runs of more than six identical nucleotides and rAOaV-1 gene-end like or gene start like sequences were removed from the computer-generated sequences by manual editing. The G/C content and the percentage of A, G, T, and C nucleotides, and of AT and GC dinucleotides, was similar between WT and codon deoptimized sequences (FIG. 1A). Percent nucleotide identity and number of nucleotide differences between WT and codon deoptimized open reading frames were less than 80%. All nucleotide changes were silent on the amino acid level. The recombinant rAOaV-1 viruses carrying individually codon deoptimized HN and F genes replicated significantly lower in chicken cells compared to rAOaV-1-WT (FIG. 1B). Notably, the recombinant rAOaV-1 carrying dual codon deoptimized HN and F genes (rAOaV-1-F.sub.CD+HN.sub.CD) were attenuated based on the plaque sizes compared to individually codon deoptimized rAOaV-1 (rAOaV-1-F.sub.CD/rAOaV-1-HN.sub.CD) or rAOaV-1-WT. The quantitative measurement of sizes of plaques in all evaluated recombinant rAOaV-1 is displayed in FIG. 1C and the difference in replication fitness is demonstrable.

[0084] Recombinant (r) rAOaV-1 were constructed using a reverse genetic system based on genotype VII strain. The rAOaV-1s were used to rescue the infectious viruses as described previously (Ayllon et al., 2013) with substantial modifications. Briefly, Vero cells were infected with modified vaccinia Ankara (MVA) expressing the T7 polymerase at a multiplicity of infection 1.0 for 6 h. These cells were transfected with Lipofectamine 2000 using rAOaVs backbones as well as supporting N, P and L gene-expression plasmids (ratio of 1:0.8:0.4:0.1) for 72 h. After 3 days post-infection, cells and cell supernatants were mixed and freeze-thawed three times at 80 C. before inoculation into 8-day-old embryonated chicken eggs. After an additional three days, individual eggs were screened using hemagglutination assay and real-time PCR as described before [OIE, 2012, Grimes, 2002, Wise et al., 2004]. Successfully rescued isolates were further propagated and purified from allantoic fluid as described previously (Kingsbury, 1966) to generate viral stock and for in vitro characterization.

EXAMPLE 2

[0085] To determine whether the codon deoptimization of F or HN ORFs individually or in combination could lead to a reduction in protein expression levels in the absence of other viral factors, DF1 cells were transfected with 250, 500, and 750 ng of plasmid and characterized for protein expression 24 h later by epifluorescence microscopy. A reduction in the fluorescent signal and the number of fluorescent cells was observed in cells transfected with codon deoptimized constructs compared with the results for cells transfected with the rAOaV-1-WT (FIG. 2A). The effects of codon deoptimization on protein expression were also tested by Western blotting (FIG. 2B). For that, the N termini of the wt, the cd construct were fused to an HA epitope tag or GFP and used to transfect DF-1 cells. Protein expression was evaluated at 24 h post-transfection using an anti-HA Mab. The pattern of expression of both proteins correlated with that previously observed for GFP-tagged constructs. While the F-.sub.WT was expressed a corresponding concentration, the F.sub.CD protein was barely detected (FIG. 2B). No such difference was observed with a loading control. Similar changes were also noticed in the HN.sub.CD genes compared to HN-.sub.WT genes (FIG. 3). Overall, these data indicate that codon deoptimization of F or HN gene reduces protein expression, which may be attributed to differences in the percentage of codon changes introduced into the viral gene, the relative quantity of the mRNAs, or a combination thereof.

EXAMPLE 3

[0086] Generation of recombinant codon-deoptimized rAOaV-1 viruses. Since F and HN constructs were efficiently expressed in transfected cells, we wanted to ascertain whether rAOaV-1 viruses encoding codon-deoptimized F and/or HN products could be rescued, as well as assess the effect of the deoptimization of viral F and/or HN products individually or in combination in the context of viral replication. To this end, codon-deoptimized viral F or HN RNA segments were incorporated into plasmid-based reverse genetics techniques in order to generate recombinant, codon-deoptimized viruses. We generated three different viruses containing codon-deoptimized synonymous mutations in coding regions comprising the entire F gene (rAOaV-1-F.sub.CD) or entire HN (rAOaV-1-HN.sub.CD) or both (rAOaV-1-F.sub.CD+HN.sub.CD). The identity of the recombinant viruses was confirmed by RT-PCR using restriction analysis and sequencing of the F and HN genes. Sequence data revealed that the F and HN gene in all recombinant viruses did not contain additional changes. Growth properties of codon-deoptimized recombinant rAOaV-1 were assessed in tissue culture. To analyse the replicative properties of recombinant codon-deoptimized viruses, we evaluated F expression levels in the context of viral infection by immunofluorescence (FIG. 4A).

EXAMPLE 4

[0087] In ovo attenuation and stability of rAOaV-1. The rAOaV-1 replicate effectively in embryonated chicken eggs. All recombinant rAOaV-1 generated in this invention were propagated in chicken eggs for 10 times at least. The replication kinetics of these recombinant viruses were determined by virus quantification assays including Western blotting (FIG. 5A), RT-PCR (FIG. 5B, C) and plaque assays (FIG. 5D, E). All viruses replicated significantly and at the comparable levels indicated that codon deoptimization is stable and doesn't pose antiviral characteristics.

EXAMPLE 5

[0088] To determine in ovo attenuation of rAOaV-1, intracerebral pathogenicity index (ICPI) was determined in 1-day-old chicks. For each ICPI test, ten 1-day-old SPF chicks were used (ten birds for test and five birds for control). The inoculum consisted of fresh, infective allantoic fluid with an HA titer for the test birds and allantoic fluid from uninfected embryonated chicken eggs for control birds. The birds were observed for clinical signs and mortality every 24 h for a period of 8 days. The scoring and determination of ICPI were done according to the method described by Alexander (1997).

[0089] In order to compare the pathogenicity of rAOaV-1-F.sub.CD, rAOaV-1-HN.sub.CD and rAOaV-1-F.sub.CD+HN.sub.CD, ICPI tests in 1-day-old chicks were performed by scoring clinical signs and mortality (Table 1). The most virulent AOaV-1 strains give indices close to 2.0, while avirulent viruses give values close to 0. In our experiments, the results of ICPI were 2 for rAOaV-1-WT, 1.18 for rAOaV-1-F.sub.CD and 1.7 for rAOaV-1-HN.sub.CD. The ICPI for rAOaV-1-F.sub.CD+HN.sub.CD was 0.0 (Table 1).

[0090] The mean death time (MDT) is hours for the minimum lethal dose to kill embryos. The minimum lethal dose is the highest virus dilution which causes all the embryos inoculated with that dilution to die. To assess MDT, 0.1 ml of the virus was inoculated into the allantoic cavity of each of five 9- to 10-day-old embryonated chicken eggs and placed in incubator at 37 C. Each egg was examined twice daily for 7 days and the times of any embryo deaths were recorded. The MDT has been used to classify rAOaV-1 strains into velogenic (taking less than 60 hours to kill), mesogenic (taking between 60-90 hours) and lentogenic (taking more than 90 hours). The MDT for rAOaV-1-WT, rAOaV-1-F.sub.CD, and rAOaV-1-HN.sub.CD was <60 hours. However, for rAOaV-1-F.sub.CD+HN.sub.CD it was >90 hours (Table 1).

[0091] The results described here show that attenuated rAOaV-1 can be used as a vaccine vector. Development of recombinant rAOaV-1 as a vaccine vector has several applications. Several foreign genes can be inserted and expressed in the same virus to obtain simultaneous immune responses to the expressed antigens in inoculated animals. For example, a single recombinant rAOaV-1 could be generated that expressed the immunogenic proteins of multiple avian pathogens or viruses of medical importance such as SARS-COV-2 (Rohaim and Munir, 2020). Alternatively, several rAOaV-1, each expressing various heterologous antigens, could be administered as a multivalent vaccine. A further extension would be to use rAOaV-1 vectors in non-avian species, where rAOaV-1 is capable of undergoing incomplete replication to the extent necessary to express inserted genes. Thus, development of rAOaV-1 as a vector should prove to be useful against avian and non-avian diseases for which suitable vaccines are not currently available.

TABLE-US-00001 TABLE 1 In ovo attenuation of codon deoptimized rAOaV-1 Intracerebral Recombinant pathogenicity virus Mean death time (MDT) index (ICPI) rAOaV-1-WT <60 hours (30 hours; exact) 2.0 rAOaV-1-F.sub.CD <60 hours (38 hours; exact) 1.8 rAOaV-1-HN.sub.CD <60 hours (38 hours; exact) 1.7 rAOaV-1- >90 hours (up to 96 hours; exact) 0.0 F.sub.CD + HN.sub.CD

EXAMPLE 6

[0092] In vivo characterization of codon-deoptimized viruses was assessed. We compared the virulence of rAOaV-1-WT and codon-optimized viruses in chicken. To ascertain whether the reduced expression of F, HN or both impacted the course of an in vivo virus infection, groups of chicken (n=10) were inoculated intranasally with 10.sup.6 or 10.sup.7 HA units and monitored for 10 days for signs of illness, weight loss, and mortality. As expected, codon-deoptimized viruses showed levels of attenuation and pathogenicity different from those for rAOaV-1-WT viruses. Animals infected with 10.sup.6 lost less body weight than infected with higher dose (10.sup.7) (FIG. 6A, B). In animals infected with 10.sup.6 or 10.sup.7, all animals infected with rAOaV-1-WT virus died within 4 or 5 days whereas mock infected animals survived. However, only 20% or 30% of animals infected with rAOaV-1-F.sub.CD virus survived at different doses. In contrast, animals infected with the same dose of rAOaV-1-HN.sub.CD virus succumbed (3% or 60%) to viral infection by day 10 (FIG. 6C, D). Interestingly, animals infected with rAOaV-1-F.sub.CD+HN.sub.CD viruses all survived as that of mock infected animals. We also evaluated the viral titers in lungs at high and low doses (FIG. 6E, F). Animals infected with rAOaV-1-F.sub.CD+HN.sub.CD showed significantly lower viral titers than animals infected with WT or individual gene codon deoptimized viruses, regardless of whether low or high dose of viruses were used. Overall, viral titres correlated with the virus dose and the degree of infection. Despite the limited attenuation observed in vitro, animals infected with rAOaV-1-F.sub.CD+HN.sub.CD virus showed less weight loss and mortality than animals infected with rAOaV-1-WT or rAOaV-1-F.sub.CD or rAOaV-1-HN.sub.CD virus.

EXAMPLE 7

[0093] Given that rAOaV-1-F.sub.CD+HN.sub.CD virus was fully attenuated in animals, we hypothesized that rAOaV-1-F.sub.CD+HN.sub.CD virus could potentially be used as a vaccine. To evaluate this possibility, chickens were vaccinated with rAOaV-1-F.sub.CD+HN.sub.CD viruses or mock vaccinated with PBS. At 7 days post-vaccination (FIG. 7A), protection was evaluated by challenging vaccinated animals with virulent AOaV (FIG. 7). Vaccinated animals with rAOaV-1-F.sub.CD+HN.sub.CD showed high antibodies (FIG. 7B) compared to mock vaccinated animals. Only animals vaccinated rAOaV-1-F.sub.CD+HN.sub.CD survived challenge (FIG. 7C) while all mock-vaccinated animals drastically lost weight and died after 3-4 days of challenge (FIG. 7D). Animal vaccinated with rAOaV-1-F.sub.CD+HN.sub.CD showed sustained antibodies levels before culling (FIG. 7E).

EXAMPLE 8

[0094] A significantly reduced tissue pathology was noticed in animals infected with rAOaV-1-F.sub.CD+HN.sub.CD viruses compared to mock-vaccinated animals (FIG. 8). These observations were observed among all organs validated by histopathological analysis exemplified here with trachea and lung. These are the important respiratory organs likely to be targeted by respiratory viruses.

[0095] The above sample embodiments should not be considered limiting to the scope of the invention whatsoever because many more embodiments and variations of embodiments are easily conceived within the teachings, scope and spirit of the instant specification.

[0096] It is to be understood that even though numerous characteristics and advantages of various embodiments of the present invention have been set forth in the foregoing description, together with the details of the structure and function of various embodiments of the invention, this disclosure is illustrative only, and changes may be made in detail, especially in matters of structure and arrangement of parts within the principles of the present invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.

[0097] It will be clear that the present invention is well adapted to attain the ends and advantages mentioned as well as those inherent therein. While presently preferred embodiments have been described for purposes of this disclosure, numerous changes may be made which readily suggest themselves to those skilled in the art and which are encompassed in the spirit of the invention disclosed and as defined in the appended claims.

REFERENCES

[0098] Krishnamurthy, S. & Samal, S. K. (1998). Nucleotide sequences of the trailer, nucleocapsid protein gene and intergenic regions of Newcastle disease virus Strain Beaudette C and completion of the entire genome sequence. Journal of General Virology 79, 2419-2424. [0099] Phillips, R. J., Samson, A. R. & Emmerson, P. T. (1998). Nucleotide sequence of the 5-terminus of New castle disease virus and assembly of the complete genomic sequence: agreement with the rule of six. Archives of Virology 143, 1993-2002. [0100] de Leeuw, O. & Peeters, B. (1999). Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the Subfamily Paramyxovirinae. Journal of General Virology 80, 131-136. [0101] Steward, M., Vipond, I. B., Millar, N. S. & Emmer son, P. T. (1993). RNA editing in Newcastle disease virus. Journal of General Virology 74, 2539-2547. [0102] Conzelmann, K.-K. (1996). Genetic manipulation of non-segmented negative-strand RNA viruses. Journal of General Virology 77, 381-389. [0103] Rmer-Oberdorfer, A., Mundt, E., Mebatsion, T., Buchholz, U. J. & Mettenleiter, T. C. (1999). Generation of recombinant lentogenic Newcastle disease virus from cDNA. Journal of General Virology 80, 2987-2995. [0104] Peeters, B. P. de Leeuw, O. S., Koch, G. & Gielkens, A. L. (1999). Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. Journal of Virol ogy 73, 5001-5009. [0105] Ayllon, J.; Garcia-Sastre, A.; Martnez-Sobrido, L (2013). Rescue of recombinant Newcastle disease virus from cDNA. JoVE, 80, e50830. [0106] OIE (2012). Newcastle Disease, Biological Standards Commission, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Mammals, Birds and Bees, 7th ed.; World Organisation for Animal Health: Paris, France, pp. 555-574. [0107] Grimes, S. E. (2002). A Basic Laboratory Manual for the Small-Scale Production and Testing of 1-2 Newcastle Disease Vaccine; FAO Regional Office for Asia and the Pacific (RAP): Bangkok, Thailand. [0108] Wise, M. G.; Suarez, D. L.; Seal, B. S.; Pedersen, J. C.; Senne, D. A.; King, D. J.; Kapczynski, D. R.; Spackman, E (2004). Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J. Clin. Microbiol. 42, 329-338. [0109] Kingsbury, D. W. (1966). Newcastle disease virus.I. Isolation and preliminary characterization of RNA from virus particles. Journal of Molecular Biology 18, 195-203. [0110] Coleman, J. Robert, Dimitris Papamichail, Steven Skiena, Bruce Futcher, Eckard Wimmer, and Steffen Mueller (2008). Virus Attenuation by Genome-Scale Changes in Codon Pair Bias. Science. June 27; 320 (5884): 1784-1787. doi: 10.1126/science. 1155761 [0111] Alexander, D. J. (1997). Newcastle disease and other avian Paramyxoviridae infections. In Diseases of Poultry, 10 edition, pp. 541-569. Edited by B. W. Calnek, Iowa State University Press, Ames, lowa. [0112] Rohaim, M, and Munir M (2020). A Scalable Topical Vectored Vaccine Candidate against SARS-COV-2. Vaccines 2020, 8 (3), 472 [0113] Weijia Wang, Xing Cheng, Paul J. Buske, JoAnn A. Suzich and Hong Jin (2019). Attenuate Newcastle disease virus by codon modification of the glycoproteins and phosphoprotein genes. Virology, 528, 144-151. DOI: https://doi.org/10.1016/j.virol.2018.12.017.