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1. WO2011092199 - MUTATED XMRV ENV PROTEINS IN THE IMMUNOSUPPRESSIVE DOMAIN

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

MUTATED XMRV ENV PROTEINS IN THE IMMUNOSUPPRESSIVE DOMAIN

The present invention relates to mutated ENV proteins and their use as vaccine.

Retroviruses are viruses, the genome of which is made up of RNA. These viruses are unique in possessing an enzyme that enables synthesis from this RNA of a DNA molecule capable of integrating into the DNA of a host cell. The retrovirus then utilizes the cell machinery to replicate. HIV is one of the best-known retroviruses. Oncogenic retroviruses (or oncoretroviruses) are cancer-causing viruses. Numerous oncoretroviruses are associated with animal diseases.

In humans, two retroviruses, called HTLV and XMRV, have been associated with a type of leukemia and with prostate cancer.

XMRV retrovirus (Xenotropic murine leukemia virus-related virus) belongs to the virus family Retro viridae and the genus gammaretro virus. It has a single-stranded RNA genome that replicates through a DNA intermediate. Its name refers to its close relationship with the murine leukemia viruses ("MuLVs"). The genome, approximately 8100 nucleotides in length, is 95% identical with several endogenous retroviruses of mice, and is 93-94% identical with several exogenous mouse viruses.

Several XMRV genomic sequences have been published to date. These sequences are almost identical, an unusual finding as retroviruses replicate their genomes with relatively low fidelity, leading to divergent viral sequences in a single host organism.

XMRV was first described in 2006 and has been isolated from human biological samples. Several reports have associated the virus with prostate cancer, and later with chronic fatigue syndrome (CFS) but other reports do not find an association. It has not yet been established whether XMRV is a cause of disease.

Report in the scientific literature [Urisman A, et al. (2006). PLoS Pathogens 2 (3): e25], as well as the international application WO 06110589, offer evidence for an association of XMRV and prostate cancer. In the initial report on XMRV, the virus was detected in cancerous prostate tissues using a microarray containing samples of genetic material from about 950 viruses. The screen indicated the presence of a gammaretro virus- like sequence in seven of eleven tumours homozygous for the R462Q mutation, but only in one of five tumours without the mutation. After isolation and cloning of the virus, an expanded screen found it present in 40% of tumours homozygous for R462Q and in only 1.5% of those not.

Additionally, a 2009 study reported XMRV infection in 23% of subjects independent of the R ase L gene variation.

Four separate studies published in December 2010 reported that three potential sources of laboratory contamination from mouse DNA casts uncertainty on the PCR evidence of XMRV positive studies [Smith RA 2010, Retrovirology 7 (1): 112].

A causal role of XMRV in cancer has not been established, and XMRV does not appear to be capable of transforming cells directly. [Metzger MJ, et al. (2010). Journal of Virology 84 (4): 1874-80]. The direct involvement of XMRV in prostate cancer remains controversial.

In prostate cancer, XMRV protein has been found in tumour-associated but nonmalignant stromal cells, but in one study is was not found in the actual prostate cancer cells, raising the possibility that the virus may indirectly support tumorigenesis [McLaughlin-Drubin ME, Munger K (2008) Biochimica et Biophysica Acta 1782 (3): 127-50]. However, in another study, XMRV proteins and nucleic acids were found in malignant cells [Aloia, et al. (2010). Cancer research, 70: 10028-10033].

In 2009, Lombardi et al. have studied blood cells from CFS (Chronic Fatigue Syndrome) patients, and identified DNA from XMRV in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls [Lombardi VC et al. (2009) Science 326 (5952): 585-9]. Experiments found that patient-derived XMRV was infectious and that transmission of the virus was possible. Secondary infections were established in uninfected lymphocytes after exposure to activated cells derived from CFS patients. These findings raised the possibility that "XMRV may be a contributing factor in the pathogenesis of CFS".

In 2010, another study examined 41 DNA samples acquired during the 1990s from 37 patients who were previously diagnosed with CFS and found MLV-like virus sequences in 32 of 37 (86.5%) compared with only 3 of 44 (6.8%) healthy donors. [Lo SC, et al. (2010) Proceedings of the National Academy of Sciences of the United States of America 107 (36): 15874-15879]. Seven of 8 patients who provided fresh blood samples almost 15 years later had both their original and their newly-drawn samples test positive. The report identified a group of MLV-related viruses, and found their sequences were more closely related to polytropic mouse endogenous retroviruses, while mouse DNA contamination was ruled out. The team concluded that further studies were needed to "determine whether the same strong association with MLV-related viruses is found in other groups of patients with CFS, whether these viruses play a causative role in the development of CFS, and whether they represent a threat to the blood supply." While addressing the issue of conflicting studies, the authors stated that their findings "clearly support" a role for MLV-like viruses in chronic fatigue syndrome and pointed out that none of the follow-up studies to date have attempted to replicate all of the original study's multiple methods used to detect XMRV.

So, to date, even if XMRV involvement in prostate cancer and CFS appear to be controversial, the eradication of XMRV appears to be a good strategy for the disease treatment.

WO2006/110589 discloses the use of the complete virus for treating prostate cancer. However, this document never demonstrates that wild type virus can be efficiently used for the vaccination of patients.

So, there is a need to provide an efficient drug in order to prevent and/or eradicate XMRV-infection associated diseases, an having an improved efficiency.

One aim of the invention is to provide a new mutated attenuated virus, having enhanced vaccinal efficiency.

Another aim of the invention is to provide a new pharmaceutical composition for treating pathologies.

The present invention relates to a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

The present invention is based on the unexpected observation made by the Inventors that mutation in the immunosuppressive domain of XMRV ENV protein both

- substantially, and in particular totally, abolish the immunosuppressive properties of

XMRV ENV protein, and

does not affect the three-dimensional structure of XMRV ENV protein, i.e. the resulting mutated XMRV ENV protein retains its structure and its antigenicity.

The minimal mutation according to the invention is a substitution of the E at position 14 of SEQ ID NO : 1 by R, H or K.

According to the invention, a given protein is said to possess an immunosuppressive property, if it is liable to inhibit the immune system of an organism in which it is present. In particular, the immunosuppressive property of said given protein can be measured by following the general procedure described in Mangeney & Heidmann (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 14920-5 and Mangeney et al. (2001) J. Gen. Virol. 82:2515-8. That is, stable tumor cell lines expressing, or in particular excreting, said given protein in the intra- or extracellular space are established and engrafted onto mice, and the size of the tumors (Aprotein) is compared, after several days, to the size of tumors (Anone) obtained from mice engrafted with tumor cell lines which do not express, or in particular excrete, said given protein. If the size of the tumors which express, or in particular excrete, the given protein is significantly greater than the size of the non-expressing, or in particular the non-excreting tumors, the given protein is said to be immunosuppressive. The immunosuppressive property of a given protein can also be characterized by its immunosuppression index [(Aprotein-Anone)/Anone]. If the immunosuppression index of a given protein is positive then the given protein is said to be immunosuppressive, and if its immunosuppression index is equal to zero or negative, the given protein is said to have essentially no immunosuppressive activity.

As intended in the present invention, the inhibiting of the immunosuppressive property of a given protein, yields a protein with substantially no immunosuppressive activity, that is having an immunosuppression index equal to zero or negative.

In one advantageous embodiment, the invention relates to a mutated XMRV ENV protein according as defined above, in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

- E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

The Inventors have demonstrated that the advantageous mutated XMRV ENV protein, harbouring the above mentioned properties, should be mutated both:

- at position 14 of SEQ ID NO : 1 , and

- at position 20 of SEQ ID NO : 1.

The above mentioned mutated XMRV ENV proteins have substantially lost, or completely lost, their immunosuppressive properties, and have a substantially enhanced, or substantially increased antigenicity compared to the antigenicity of the corresponding wild type XMRV ENV protein.

The above mentioned mutated XMRV ENV proteins have substantially lost, or lost, their immunosuppressive properties, and have a substantially enhanced, or increased antigenicity compared to the antigenicity of the corresponding wild type XMRV ENV protein.

The antigenicity of said mutated XMRV ENV proteins is at least 5 fold, preferably at least 10 fold higher compared to the antigenicity of the wild type XMRV ENV protein.

In another advantageous embodiment, the invention relates to the mutated XMRV ENV previously defined, wherein said two mutations are such that

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F, M, Y or W.

Advantageous mutated XMRV ENV proteins according to the invention are proteins wherein: - E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and A which is at the position 20 of SEQ ID NO: 1 is substituted by F,

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and A which is at the position 20 of SEQ ID NO: 1 is substituted by M,

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and A which is at the position 20 of SEQ ID NO: 1 is substituted by Y,

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and A which is at the position 20 of SEQ ID NO: 1 is substituted by Y,

E which is at the position 14 of SEQ ID NO : 1 is substituted by H, and A which is at the position 20 of SEQ ID NO: 1 is substituted by F,

- E which is at the position 14 of SEQ ID NO : 1 is substituted by H, and A which is at the position 20 of SEQ ID NO: 1 is substituted by M,

E which is at the position 14 of SEQ ID NO : 1 is substituted by H, and A which is at the position 20 of SEQ ID NO: 1 is substituted by Y,

E which is at the position 14 of SEQ ID NO : 1 is substituted by H, and A which is at the position 20 of SEQ ID NO: 1 is substituted by Y,

E which is at the position 14 of SEQ ID NO : 1 is substituted by K, and A which is at the position 20 of SEQ ID NO: 1 is substituted by F,

- E which is at the position 14 of SEQ ID NO : 1 is substituted by K, and A which is at the position 20 of SEQ ID NO: 1 is substituted by M,

E which is at the position 14 of SEQ ID NO : 1 is substituted by K, and A which is at the position 20 of SEQ ID NO: 1 is substituted by Y, and

E which is at the position 14 of SEQ ID NO : 1 is substituted by K, and A which is at the position 20 of SEQ ID NO: 1 is substituted by Y.

In another advantageous embodiment, the invention relates to the mutated XMRV ENV previously defined, wherein said two mutations are such that

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F.

In another advantageous embodiment, the invention relates to the mutated XMRV ENV previously defined, wherein wild type XMRV ENV protein consists of the amino acid sequences chosen among the group comprising: SEQ ID NO : 2, (YP_512363.1), SEQ ID NO : 3, (ABB83229.1), SEQ ID NO : 4, (ACY30457.1), SEQ ID NO : 5, (ABM47429.1), SEQ ID NO : 6, (ACY3046.1), SEQ ID NO : 7, (ABB83226.1), and SEQ ID NO : 8, (ACY30462.1).

In another advantageous embodiment, the invention relates to the mutated XMRV ENV previously defined, wherein said mutated XMRV ENV protein consists of the amino acid sequence chosen among the group comprising: SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22 and SEQ ID NO : 23.

The invention also relates to a nucleic acid molecule coding for mutated XMRV ENV protein as defined above.

The invention also relates to a nucleic acid molecule coding for mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLCAA),

said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

In one advantageous embodiment, the invention relates to the nucleic acid molecule as defined above, in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

- E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

In one advantageous embodiment, the invention relates to the nucleic acid molecule as defined above, wherein said two mutations are such that

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F, M, Y or W.

In one advantageous embodiment, the invention relates to the nucleic acid molecule as defined above, wherein said two mutations are such that

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F.

In one another advantageous embodiment, the invention relates to the nucleic acid molecule as defined above, wherein wild type XMRV ENV protein consists of the amino acid sequence chosen among the group comprising: SEQ ID NO : 2, (YP_512363.1), SEQ ID NO : 3, (ABB83229.1), SEQ ID NO : 4, (ACY30457.1), SEQ ID NO : 5, (ABM47429.1), SEQ ID NO : 6, (ACY3046.1), SEQ ID NO : 7, (ABB83226.1), and SEQ ID NO : 8, (ACY30462.1).

In another advantageous embodiment, the invention relates to the nucleic acid molecule as defined above coding for a mutated XMRV ENV protein consisting of the amino acid sequence chosen among the group comprising: SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16,

SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22 and SEQ ID NO : 23.

The invention also relates to a vector comprising a nucleic acid molecule as defined above, coding for a mutated XMRV ENV as defined above.

The invention also relates to a vector comprising a nucleic acid molecule coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

In one advantageous embodiment, the invention relates to the vector previously defined, coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

In one advantageous embodiment, the invention relates to the vector previously defined, coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F, M, Y or W.

In one advantageous embodiment, the invention relates to the vector previously defined, coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F.

In one advantageous embodiment, the invention relates to the vector previously defined, coding for a mutated XMRV ENV protein consisting of the amino acid sequence chosen among the group comprising: SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 1 1, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22 and SEQ ID NO : 23.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising nucleic acid sequences allowing the expression of said nucleic acid molecule, as defined above, said nucleic acid molecule coding for a mutated XMRV ENV protein as defined above.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising nucleic acid sequences allowing the expression of at least one another XMRV protein.

In one advantageous embodiment, the invention relates to the vector previously defined, in association with at least one another vector as defined above comprising nucleic acid sequences allowing the expression of at least one another XMRV protein.

According to the invention other XMRV proteins can be GAG, PRO and POL XMRV proteins.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising nucleic molecule coding for XMRV GAG protein.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising nucleic acid molecule coding for XMRV POL protein.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising nucleic acid molecule coding for XMRV PRO protein.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising a first nucleic acid molecule coding for XMRV GAG protein, and a second nucleic acid molecule coding for XMRV POL protein.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising a first nucleic acid molecule coding for XMRV GAG protein, and a second nucleic acid molecule coding for XMRV PRO protein.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising a first nucleic acid molecule coding for XMRV PRO protein, and a second nucleic acid molecule coding for XMRV POL protein.

In one advantageous embodiment, the invention relates to the vector previously defined, further comprising a first nucleic acid molecule coding for XMRV GAG protein, a second nucleic acid molecule coding for XMRV PRO protein, and a third nucleic acid molecule coding for XMRV POL protein.

In one advantageous embodiment, the invention relates to the vector previously defined, said vector being a viral vector, in particular a pox vector chosen among a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a measles vector, or a CMV (cytomegalovirus) vector.

In one advantageous embodiment, the invention relates to the vector previously defined, in association with at least one another vector, said another vector being a viral vector, in particular a pox vector chosen among a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a measles vector, or a CMV (cytomegalovirus) vector, comprising nucleic acid sequences allowing the expression of at least one another XMRV protein.

According to the invention other XMRV proteins can be GAG, PRO and POL XMRV proteins.

Measles vector as described in Combredet, C, et al. 2003. J. Virol. 77 (21), 11546-11554 and Guerbois, M., (2009) Virology 388 191-203, can be used as vectors according to the invention.

Canarypox vector as described in Poulet, H., et al (2003) Veterinary Record; 153:141-145 can be used as vector according to the invention.

Adenovirus vectors as described in Bayer, W., et al. (2008) Vaccine 26, 716-726, and in particular Bayer, W., et al. (2010) J. Virol. 84, 1967-1976, can be used as vector according to the mvention.

The skilled person knows how to use the vectors disclosed above and hereafter in order to insert mutated XMRV ENV protein according to the invention, and possibly at least one another XMRV protein.

In one advantageous embodiment, the invention relates to the vector previously defined, said vector being a viral vector, wherein in particular a pox vector chosen among a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a measles vector, or a CMV (cytomegalovirus) vector.

The vector mentioned above comprises:

nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

nucleic acid molecule coding for another XMRV protein, which is not ENV protein.

In one advantageous embodiment, the invention relates to the vector previously defined, said vector being a viral vector, wherein in particular a pox vector chosen among a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a measles vector, or a CMV (cytomegalovirus) vector.

The vector mentioned above comprises:

nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

nucleic acid molecule coding for GAG XMRV protein.

Advantageous vectors are Measles vector as described in Combredet, C, et ai. 2003. J. Virol. 77 (21), 11546-1 1554 and Guerbois, M., (2009) Virology 388 191-203, and canarypox vector as described in Poulet, H., et ai. (2003) Veterinary Record; 153:141-145.

In one advantageous embodiment, the invention relates to the vector previously defined, said vector being a viral vector, wherein in particular a pox vector chosen among a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a measles vector, or a CMV (cytomegalovirus) vector.

The vector mentioned above comprises:

nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

nucleic acid molecule coding for PRO XMRV protein.

In one advantageous embodiment, the invention relates to the vector previously defined, said vector being a viral vector, wherein in particular a pox vector chosen among a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a measles vector, or a CMV (cytomegalovirus) vector.

The vector mentioned above comprises:

- nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

nucleic acid molecule coding for POL XMRV protein.

In one advantageous embodiment, the invention relates to the vector previously defined, said vector being a viral vector, wherein in particular a pox vector chosen among a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a measles vector, or a CMV (cytomegalovirus) vector.

The vector mentioned above comprises:

- nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

nucleic acid molecule coding for GAG XMRV protein, and

nucleic acid molecule coding for PRO XMRV protein.

The vector mentioned above comprises:

nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

- nucleic acid molecule coding for GAG XMRV protein, and

nucleic acid molecule coding for POL XMRV protein.

The vector mentioned above comprises:

nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

nucleic acid molecule coding for PRO XMRV protein, and

nucleic acid molecule coding for POL XMRV protein.

The vector mentioned above comprises:

- nucleic acid molecule coding for mutated XMRV ENV protein according to the invention, and

nucleic acid molecule coding for GAG XMRV protein, and

nucleic acid molecule coding for PRO XMRV protein, and

nucleic acid molecule coding for POL XMRV protein.

The invention also relates to a composition, in particular a pharmaceutical composition, or a vaccinal composition, preferably in association with a pharmaceutically acceptable carrier or vehicle, comprising:

a mutated XMRV ENV protein, as defined above, or

- a nucleic acid molecule coding for a mutated XMRV ENV protein, as defined above, or a vector, as defined above, or

or a combination of the above.

The invention also relates to a composition, in particular a pharmaceutical composition, or a vaccinal composition, as defined above, for its use as medicine or drug or vaccine.

The appropriate dosage of the composition of the invention can be adapted as a function of various parameters, in particular the mode of administration; the composition employed; the age, health, and weight of the host organism; the nature and extent of symptoms; kind of concurrent treatment; the frequency of treatment; and/or the need for prevention or therapy. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by a practitioner, in the light of the relevant circumstances.

A composition based on vector plasmids may be administered in doses of between 10 μg and 20 mg, advantageously between 100 μg and 2 mg. A protein composition may be administered in one or more doses of between 10 ng and 20 mg, advantageously a dosage from about 0.1 μg to about 2 mg of the therapeutic protein per kg body weight. The administration may take place in a single dose or a dose repeated one or several times after a certain time interval.

The invention also relates to a composition, in particular a pharmaceutical composition, or a vaccinal composition, preferably in association with a pharmaceutically acceptable carrier or vehicle, comprising a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

As used herein, a "pharmaceutically acceptable vehicle" is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, compatible with pharmaceutical administration.

In one advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, comprising a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA),

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

In one another advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, comprising a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F, M, Y or W.

In one another advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA),

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F.

In one another advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, wherein wild type XMRV ENV protein consists of the amino acid sequence chosen among the group comprising: SEQ ID NO : 2, (YP_512363.1), SEQ ID NO : 3, (ABB83229.1), SEQ ID NO : 4, (ACY30457.1), SEQ ID NO : 5, (ABM47429.1), SEQ ID NO : 6, (ACY3046.1), SEQ ID NO : 7, (ABB83226.1), and SEQ ID NO : 8, (ACY30462.1).

In one another advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, wherein said mutated XMRV ENV protein consists of the amino acid sequence chosen among the group comprising SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22 and SEQ ID NO : 23.

The invention also relates to a composition, in particular a pharmaceutical composition, or a vaccinal composition, preferably in association with a pharmaceutically acceptable carrier or vehicle, comprising a nucleic acid molecule coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO: 1, (LQNRRGLDILFLKEGGLC AA) , said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

In one advantageous embodiment the invention relates to the composition, in particular a pharmaceutical composition, defined above, comprising a nucleic acid molecule coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO: 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

In one advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, comprising a nucleic acid molecule coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO: 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F, M, Y or W.

In one advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, comprising a nucleic acid molecule coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO: 1, (LQNRRGLDILFLKEGGLC AA) ,

said two mutations being such that :

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, and

- A which is at the position 20 of SEQ ID NO: 1 is substituted by F.

In one advantageous embodiment, the invention relates to the composition, in particular a pharmaceutical composition, defined above, wherein wild type XMRV ENV protein consists of the amino acid sequence chosen among the group comprising: SEQ ID NO : 2, (YP_512363.1), SEQ ID NO : 3, (ABB83229.1), SEQ ID NO : 4, (ACY30457.1), SEQ ID NO : 5, (ABM47429.1), SEQ ID NO : 6, (ACY3046.1), SEQ ID NO : 7, (ABB83226.1), and SEQ ID NO : 8, (ACY30462.1).

In one advantageous embodiment, the invention relates to the composition defined above, wherein said mutated XMRV ENV protein consists of the amino acid sequence chosen among the group comprising: SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22 and SEQ ID NO : 23.

The invention also relates to the use of

a mutated XMRV ENV protein, as defined above, or

- a nucleic acid coding for a mutated XMRV ENV protein, as defined above, or

a vector comprising nucleic acid molecule coding for a mutated XMRV ENV protein, as defined above, or

a combination of the above, or

a composition as defined above,

for a preparation of a drug intended for the prevention and/or the treatment of pathologies involving the XMRV virus, in particular for the prevention or the treatment of prostate cancer and chronic fatigue syndrome.

The invention also relates to a composition comprising:

a mutated XMRV ENV protein, as defined above, or

a nucleic acid coding for a mutated XMRV ENV protein, as defined above, or a vector comprising nucleic acid molecule coding for a mutated XMRV ENV protein, as defined above, or

a combination of the above,

a composition as defined above,

for its use for the prevention and/or the treatment of pathologies involving the XMRV virus, in particular for the prevention or the treatment of prostate cancer and chronic fatigue syndrome.

The invention also relates to a method for the prevention or treatment of XMRV infection, comprising the administration in a person in a need thereof of a therapeutically effective amount of a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

In one advantageous embodiment, the invention relates to the method for the prevention or treatment of XMRV infection, as defined above, comprising the administration in a person in a need thereof of a therapeutically effective amount of a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations , said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA),

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

In one advantageous embodiment, the invention relates to the method for the prevention or treatment of XMRV infection, as defined above, comprising the administration in a person in a need thereof of a therapeutically effective amount of a nucleic acid molecule coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation, said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

In one advantageous embodiment, the invention relates to the method for the prevention or treatment of XMRV infection, as defined above, comprising the administration in a person in a need thereof of a therapeutically effective amount of a nucleic acid coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations , said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

- E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

The invention also relates to a method for the prevention or treatment of XMRV infection, comprising the administration in a person in a need thereof of a therapeutically effective amount of vector comprising a nucleic acid molecule coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents at least one mutation , said immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKEGGLC AA) ,

said at least one mutation being such that E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K.

The invention also relates, in one another advantageous embodiment, to the method for the prevention or treatment of XMRV infection, as defined above comprising the administration in a person in a need thereof of a therapeutically effective amount of a vector comprising a nucleic acid coding for a mutated XMRV ENV protein in which the immunosuppressive domain of the wild type XMRV ENV protein presents two mutations, said

immunosuppressive domain of the wild type XMRV ENV protein consisting of the amino acid sequence SEQ ID NO : 1, (LQNRRGLDILFLKE GGLC AA) ,

E which is at the position 14 of SEQ ID NO : 1 is substituted by R, H or K and

A which is at the position 20 of SEQ ID NO: 1, is such that it ensures that the structure of the viral XMRV ENV protein is conserved.

All compositions mentioned above can be administered in a host via different routes: intraperitoneal (i.p.), subcutaneous (s.c), intradermal (i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, oral administration and intranasal administration or inhalation.

If desired, the method of the invention can be carried out in conjunction with one or more conventional therapeutic modalities (e.g. radiation, chemotherapy and/or surgery). The use of multiple therapeutic approaches provides the patient with a broader based intervention. In one embodiment, the method of the invention can be preceded or followed by a surgical intervention. In another embodiment, it can be preceded or followed by radiotherapy (e.g. gamma radiation). Those skilled in the art can readily formulate appropriate radiation therapy protocols and parameters which can be used (see for example Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations and modifications as will be readily apparent to those skilled in the field).

The invention will be better illustrated by the following figures and the example, without limiting its scope.

FIGURE LEGENDS

Figures 1A-D : Characterization of the fusogenic and IS activities of F-MLV Env and generation of a fusion-positive immunosuppression-negative specific mutant.

Figure 1A is a schematic representation of the F-MLV Env with the surface (SU) and transmembrane (TM) subunits, the furin cleavage site, the hydrophobic fusion peptide, the transmembrane anchor, and the IS domain with its peptide sequence indicated; the E>R and A>F substitutions generated in the single or double mutants are positioned.

Figure IB represents the infectivity of Mo-MLV virions pseudotyped with F-MLV Env, wild-type (first column), single mutant (E14R second column, A20F third column), or

double-mutant (DM fourth column). Infectivity is measured using NIH/3T3 cells as target cells and is expressed as LacZ-positive focus forming units (ffu) per ml of supernatant (mean +/- SD, n=3). Y axis is a logarithmic representation of the infectivity (ffu/mL).

Figure 1C represents the w vivo immunosuppressive (IS) activity of WT (first column) and DM (second column) F-MLV Env. IS activity is assayed using F-MLV env-transduced MCA205 tumor cells engrafted into allogenic BALB/c mice, and quantified by an index based on tumor size (see Results and [Mangeney M, et al. (2007) Proc Natl Acad Sci U S A 104:20534-9]; mean +/- SD, n=3). Y axis represents the immunosuppressive index.

Figure ID represents a curve comparing the in vitro propagation rates of WT (black circles) and DM (grey circles) Env-containing F-MLV virions, using NIH/3T3 cells as target cells. Viral loads in supernatants are measured by qRT-PCR (mean +/- SD, n=4). Y- axis represents the number of virus in supernatant (RNA copy/mL), and X- axis represents hours after infection.

Figures 2A and B represent the requirement of the immunosuppression-positive domain for F-MLV propagation in immunocompetent mice.

Viremia of untreated or X-ray-irradiated Swiss mice upon infection with WT (black circles) or the non-immunosuppressive DM (grey circles) F-MLV. Mice were intra veinously. injected at day 0, and viral loads were quantified by qRT-PCR from blood samples collected at the indicated times (viral RNA copies/mL).

Each circle corresponds to one individual mouse, with the lines connecting the geometric means. No viral RNA was detected in PBS-injected control mice.

Figure 2A represents results obtained with untreated mice.

Figure 2B represents results obtained with mice that were X-ray-irradiated (5 Gray) 2 days before infection.

Figure 3: Cellular targets of the Env-mediated immunosuppression effect, assayed by immune cell depletions in vivo. Serum viral loads after injection of WT (circles) or DM (squares) F-MLV were measured in untreated (closed symbols) or NK.1.1 -depleted (open symbols) Swiss (A) or Swiss-Nude (B) mice, or in CD8-depleted (closed symbols) or CD8-depleted plus NK1.1 -depleted (open symbols) Swiss mice (C). The data correspond to the mean +/- SD for five mice, and are representative of 2-7 independent experiments. Cell depletions (duration of antibody treatment indicated with a bar, see Methods) were controlled by flow cytometry on blood samples (upper panels), with numbers in the dot plot corners indicating the percentage of labeled cells in the corresponding quadrant. NK cell recovery after the last antibody injection was 50% 5 days later and >90% at day 8.

Figure 4A and B represent the protective activity and the enhanced immunogenicity conferred by mutation within the F-MLV ISD.

Figure 4A represents a curve showing the protection of mice immunized with DM F-MLV and challenged with WT F-MLV. Swiss mice were injected with 8 x 108 RNA copies of DM F-MLV (white and grey circles) or injected with PBS (black circles), and the absence of viremia was checked four weeks later (day -35). Nine weeks post infection, mice were challenged (indicated by arrow) with 4 x 107 RNA copies of WT F-MLV (grey and black circles) or injected with PBS (white circles), and post-challenge sera were collected at the indicated time points. Each circle corresponds to one individual mouse, with the lines connecting the geometric means of 8 mice; data are representative of more than (>) 3 independent experiments, with in all cases no significant departure from full control of viremia by the vaccinated mice. Y- axis represents the viral load (RNA copies/mL) and X-axis represents the days after infection

Figure 4B represents graphs comparing immunogenicity (B-cell responses) of WT and DM UV-inactivated F-MLV. C57B1/6 mice were injected thrice at one week interval with 109 RNA copies of UV-inactivated WT or DM F-MLV in the presence of 50 μg of CpG ODN. (B) One week after the last injection, mice were blood sampled and serially diluted serum was used to detect TM-specific (left) and Gag-specific (right) IgG by ELISA. Results are the mean +/- SD of five mice, and are representative of 3 independent experiments.

Figure 4C represents graphs comparing immunogenicity (T-cell responses) of WT and DM UV-inactivated F-MLV. C57B1/6 mice were injected thrice at one week interval with 109 RNA copies of UV-inactivated WT or DM F-MLV in the presence of 50 μg of CpG ODN. Ten days after the last injection, mice were sacrificed and splenocytes restimulated in vitro for 72 h in the presence or absence of the I-Ab restricted HI 9 Env peptide (left) or Kb-restricted Gag peptide (right). Specific IFN-γ secretion by CD4 or CD8 T cells was detected in culture supernatants by standardized sandwich ELISA. Data are the mean +/- SD of three independent experiments performed with 3-4 mice per group.

Figures 5A to G represents the ex vivo viremia of WT and DM GFP-marked F-MLV. GFP marked F-MLV virions were generated by transient transfection of 293T cells with p57-IRESgfp plasmids, and the filtered supematants were used to infect NIH/3T3 cells under conditions similar to those in Figure 1.

Figure 5A represents phase-contrast microscopy visualization GFP-expressing cells infected with no virus (mock transfected 293T-cell supernatant) 4 days postinfection.

Figure 5B represents phase-contrast microscopy visualization GFP-expressing cells infected with DM GFP-marked F-MLV, 4 days postinfection.

Figure 5C represents phase-contrast microscopy visualization GFP-expressing cells infected with WT GFP-marked F-MLV 4 days postinfection.

Figure 5D represents fluorescence microscopy visualization GFP-expressing cells infected with no virus (mock transfected 293T-cell supernatant) 4 days postinfection.

Figure 5E represents fluorescence microscopy visualization GFP-expressing cells infected with DM GFP-marked F-MLV, 4 days postinfection.

Figure 5F represents fluorescence microscopy visualization GFP-expressing cells infected with WT GFP-marked F-MLV 4 days postinfection.

Figure 5G represents a curve comparing in vitro propagation kinetics of WT (black circles) and DM (gray circles) GFP-marked F-MLV. The percentages of GFP+ infected NIH/3T3 cells were determined by flow cytometry using a FACScalibur flow cytometer (BD Biosciences) at days 1 , 4, and 6 postinfection (mean ± SD of three independent experiments). Y-axis represents the percentage of infected cells, and X-axis represnts the days post infection.

Figure 6 : Control injection of anti-NKl .l antibodies into NK1.1- Swiss mice does not allow mutant F-MLV to propagate. (Upper) Phenotyping of NK1.1+ and NK1.1- Swiss mice as visualized by flow cytometry on blood samples using anti-NKl . l and anti-NKG2A,C,E antibodies. (Lower) Serum viral loads of mice after injection of WT (·) or immunosuppression-negative DM (□ and■) F-MLV as measured in untreated Swiss mice (· and■) or in NK.1.1- Swiss mice injected with the anti-NKl . l antibody (□). Each symbol corresponds to the mean ± SD of more than three mice. PE means phycoerythrin.

Figure 7: Natural Treg cells do not affect F-MLV viremia. Swiss mice were injected with 100 μg of anti-CD25 IgG on day -1 (Materials and Methods) and were infected at day 0 with 4 χ 107 copies of WT or DM F-MLV. (Upper) Cell depletion was checked by flow cytometry on

blood samples 2 days postinfection. Numbers in the dot plot corners indicate the percentage of labeled cells in the corresponding quadrant. (Lower) Serum viral loads after injection of WT (o and ·) or DM (□ and■) F-MLV was detected in untreated (· and■) or CD25-depleted (o and □) Swiss mice. Each circle corresponds to one individual mouse, with the lines connecting the geometric means, and results are representative of three independent experiments. PE means phycoerythrin.

Figures 8A to D represent curves analysing the antiviral antibody responses to WT and DM F-MLV [and control (PBS)]. Swiss mice were i.v. inoculated as in Figure 2 with 2 x 107 copies of WT or DM F-MLV, and serum was harvested 8 weeks postinfection.

Figure 8A represents ELISA results obtained with Serially diluted serum to detect SU specific IgG. Each symbol represents one individual mouse with the bars corresponding to the means, and results are representative of three independent experiments. Y- axis represents amount of IgG (ng/niL).

Figure 8B represents ELISA results obtained with Serially diluted serum to detect TM specific IgG. Each symbol represents one individual mouse with the bars corresponding to the means, and results are representative of three independent experiments. Y- axis represents amount of IgG (ng/niL).

Figure 8C represents ELISA results obtained with Serially diluted serum to detect TM specific IgG. Each symbol represents one individual mouse with the bars corresponding to the means, and results are representative of three independent experiments. Y- axis represents amount of IgG (arbitrary units; a.u.).

Figure 8D represents assay for F-MLV neutralizing antibodies in the collected sera. Neutralizing antibodies were tested for their ability to inhibit infection of NIH/ 3T3 target cells by F-MLV. Results for serum of WT (·) or DM (o) F-MLV-infected mice are expressed as percentage of integrated proviral copies relative to controls with serum from mock-infected (with PBS) mice. Each symbol corresponds to the mean of triplicates ± SD for each individual mouse. Y-axis represents F-MLV provirus integration in percent, and X- axis indicates the sera dilutions.

Figures 9 A and B represent functional immunosuppressive domains in human retrovirus Envs.

Figure 9A represents sequence alignment of the F-MLV, XMRV, and HTLV-1 retrovirus and of the syncytin-1 (Syn- 1) ectodomains and identification of the two residues controlling their IS activity.

Figure 9B represents histograms showing the differential antibody responses induced by WT and DM recombinant ectodomains. Recombinant proteins corresponding to the 64 amino acids (as shown in figure 9A) of the WT or DM TM ectodomain for F-MLV, XMRV, and Syn-1 and to the 84 amino acids of the WT or DM TM ectodomain fused with the MBP protein for HTLV-1 were i.v. injected three times with a 1-week interval into Swiss mice (50 μg per injection). One week after the last injection, mice were blood-sampled and IgG levels were determined by ELISA using plates coated with the appropriate WT ectodomain protein for F-MLV, XMRV, and Syn-1 or with MBP-LacZ for HTLV-1. IgG levels are expressed relative to that of the WT ectodomain set as unity for retroviruses (and to that of the DM ectodomain for syncytin-1). Results are representative of 2-5 independent experiments. Similar results were observed when ELISA plates were coated with the DM ectodomain proteins from F-MLV, XMRV, and Syn-1. Y- axis represents amount of antibodies expressed in arbitrary units (a.u).

IS activity (+ or -) of the corresponding ectodomains as measured by the MCA205 tumor rejection assay in Figure 1C, are indicated under the histograms. MCA205 tumor cells were transduced with pDFG expression vectors encoding either the WT or DM version of secreted F-MLV, XMRV, HTLV-1, and Syn-1 ectodomains. The tumor rejection assay was performed as described in Materials and Methods.

EXAMPLES

Example 1

As both clinical and experimental data have shown, most viruses have adapted to prevent their rejection by the host immune system during acute infection, to an extent resulting sometimes in the establishment of persistent infections [Bowie AG and Unterholzner L (2008) Nat Rev Immunol 8:911-22; Hansen TH, Bouvier M (2009). Nat Rev Immunol 9:503-13; Jonjic S et al. (2008) Curr Opin Immunol 20:30-8; Malim MH and Emerman M (2008) Cell Host Microbe 3:388-98]. Persistent viruses, and notably retroviruses, have developed diverse strategies to subvert the host antiviral response, which allow them to escape the innate and adaptive immune system, by directly affecting and/or inducing specific sets of immune cells, including NK, CD8 T or regulatory T (Treg) cells [lannello A, et al. (2006) J Leukoc Biol 79:16-35; Schneider-Schaulies S and Dittmer U (2006) J Gen Virol 87:1423-38]. To characterize -and possibly counteract - the mechanisms by which retroviruses escape immune rejection, the Inventors thought that identification of the viral effector protein(s) associated with this activity was an unavoidable pre-requisite.

An immunosuppressive (IS) activity of retroviral envelope proteins (Envs) was initially reported ex vivo [Cianciolo G, et al. (1985) Science 230:453-455] and further substantiated in the Inventors group using an in vivo tumor rejection assay [Blaise S, et al. (2001) J. Gen. Virol. 82:1597-1600; Mangeney M and Heidmann T (1998) Proc. Natl. Acad. Sci. U. S. A. 95:14920-14925; Mangeney M, et al. (2007) Proc Natl Acad Sci US A 104:20534-9]. In this model, the Inventors showed that the full-length Env of Moloney Murine Leukemia Virus (Mo-MLV) as well as its transmembrane (TM) subunit alone are immunosuppressive [Mangeney M and Heidmann T (1998) Proc. Natl. Acad. Sci. U. S. A. 95:14920-14925], and further delineated a highly conserved immunosuppressive domain (ISD) within retroviral Envs [Mangeney M, et al. (2007) Proc Natl Acad Sci U S A 104:20534-9]. However, the importance of this IS function for infectious retrovirus propagation in vivo remained unknown.

To answer this question, it was

i) looked for a murine retrovirus whose viremia can be easily monitored in vivo, and ii) attempted to introduce appropriate mutations into a cloned element so as to very specifically knockout the IS activity of the viral Env, without altering its fusogenic properties nor the overall replicative properties of the retrovirus in ex vivo assays or in immunocompromised mice.

The Friend Virus (FV) is a complex of mouse retroviruses composed of the non-pathogenic replication-competent Friend Murine Leukemia Virus (F-MLV) and a pathogenic replication-defective spleen focus-forming virus (SFFV). An F-MLV provirus has been cloned [Sitbon M, et al. (1990) J Virol 64:2135-40], making possible ex vivo production of N-tropic infectious particles, as well as introduction of definite mutations within the viral sequence by genetic engineering. F-MLV was therefore selected as a suitable model for the present in vivo studies.

In previous work on the ISD of the captured endogenous retroviral syncytin proteins [Mangeney M, et al. (2007) Proc Natl Acad Sci U S A 104:20534-9], and owing to the unexpected characteristic of one of them (syncytin-1) which has lost its IS activity but has fully conserved its "mechanical" fusogenic activity, the Inventors were able to identify, within the ISD, definite amino-acids (aa) that govern IS activity, as well as specific amino acid (aa) substitutions that allowed its switching on and off, without altering fusogenicity of the mutated Envs.

Owing to the incredibly high structure conservation within the ectodomains of retroviral Envs [Renard M, et al. (2005) J Mol Biol 352:1029-34], the Inventors have here attempted to generate a mutant F-MLV deprived of IS activity, by following the same rules. This led us to construct an F-MLV immunosuppression-negative mutant, with infectious capacity indistinguishable from that of the wild type virus in vitro. This mutant allowed us to unambiguously demonstrate that i) a functional ISD is absolutely required for viremia in vivo, in immunocompetent mice, ii) the ISD inhibits both the innate and adaptive arms of the immune response, and iii) the mutations within the ISD resulting in loss of its IS activity are associated with enhanced immunogenicity of the viral antigens. The Inventors also identified functional ISDs in other retroviruses, namely the human T cell leukemia virus (HTLV) and the xenotropic MLV related virus (XMRV) initially discovered in human prostate tumors [ Urisman A, et al. (2006) PLoS Pathog 2:e25], which therefore become, owing to the present mouse model and experimental results, definite targets for therapeutic antiviral approaches, as well as optimized vaccine antigens when adequately mutated.

Generation of a mutant, immunosuppression-negative, functional Friend Murine Leukemia Virus.

As illustrated in Figure 1A, the MLV retroviral Env comprises within its TM subunit a sequence that the Inventors have previously delineated as responsible for its IS activity. The 20 aa sequence shown in Figure 1A corresponds to that of both Mo- and F-MLV. Despite

significant divergence between the primary sequence of the 20 aa ISD of MLV Env and that of the previously characterized syncytin-1 and Mason-Pfizer monkey virus (MPMV) Env, the Inventors reasoned that the high 3D-structure conservation of ISDs [Mangeney M, et al. (2007) Proc Natl Acad Sci USA 104:20534-9; Renard M, et al. (2005) J Mol Biol 352: 1029-34] should allow a direct identification of the aa involved in IS activity as well as the substitutions required for specific loss of IS function within MLV ISD. Accordingly, the Inventors replaced the key E14 and A20 residues of the F-MLV Env ISD by those of the non-immunosuppressive syncytin-1, namely R14 and F20 (Figure 1A) as described in the Inventors previous work [Mangeney M, et al. (2007) Proc Natl Acad Sci USA 104:20534-9], and checked whether it would affect infectivity of Env pseudotypes (Figure IB). As previously described for MPMV Env [Mangeney M, et al. (2007) Proc Natl Acad Sci U S A 104:20534-9], the single mutation of one or the other of these two key residues altered -although not to the same extent- infectivity, but the double mutation did not, showing that combining the E14R and A20F mutations in the double mutant (DM) fully restores infectivity to wild-type (WT) level.

The immunosuppressive property of DM F-MLV Env was then analyzed using the in vivo tumor rejection assay that the Inventors had previously used to demonstrate IS activity of the MoMLV and MPMV Env, as well as of the MoMLV TM subunit alone [Blaise S, et al. (2001) J. Gen. Virol. 82:1597-1600; Mangeney M, Heidmann T (1998) Proc. Natl. Acad. Sci. U. S. A. 95:14920-14925; Mangeney M, et al. (2007) Proc Natl Acad Sci USA 104:20534-9]. The rationale of the assay has been previously described [Mangeney M, Heidmann T (1998) Proc. Natl. Acad. Sci. U. S. A. 95:14920-14925; Mangeney M, et al. (2007) Proc Natl Acad Sci U S A 104:20534-9] and can be summarized as follows: while injection of MCA205 tumor cells (H-2b) into allogeneic Balb/c mice (H-2d) leads to the formation of no tumor or transient tumors that are rapidly rejected, injection of the same cells, but stably expressing an immunosuppressive retroviral Env, leads to the growth of larger tumors that persist for a longer time - in spite of the expression of the new exogenous antigen. This difference is not associated with a difference in intrinsic cell growth rate since it is not observed in syngeneic C57BL/6 mice, and is immune system-dependent [Mangeney M, Heidmann T (1998) Proc. Natl. Acad. Sci. U. S. A. 95:14920-14925; Mangeney M, et al. (2007) Proc Natl Acad Sci US A 104:20534-9]. The extent of "immunosuppression" can be quantified by an index based on tumor size, a positive index indicating that env expression facilitates tumor growth, as a consequence of its IS activity, a null or negative index pointing to no effect or even enhanced rejection, respectively (the latter may be explained by a stimulation of the immune response of the host against the new foreign antigen, represented by a non-immunosuppressive Env, expressed at the surface of the tumor cells). As illustrated in Figure 1C, and as expected from the 100% sequence identity between the Mo- and F-MLV ISD, F-MLV Env is immunosuppression-positive and, remarkably, the doubly mutated, but still functional, Env becomes immunosuppression-negative.

The Inventors then replaced the WT env gene by its non-immunosuppressive DM counterpart in the F-MLV proviral molecular clone 57 [Sitbon M, et al. (1990) J Virol 64:2135-40], and produced ex vivo each type of retroviral particles. Viral particles were generated upon transfection of 293T cells with the WT or DM p57 plasmid and infection of NIH/3T3 producer cells with the harvested cell supernatant. Virus yields for either plasmid were similar as measured by a quantitative RT-PCR assay of viral RNA in the NIH/3T3 producer cell supernatants. Furthermore, both viruses display the same propagation kinetics in an in vitro infection assay in NIH/3T3 cells (Figure ID; see also Figures 5 for IRES-g p-marked viruses), confirming that F-MLV DM Env is fully functional and that the introduced mutations have no effect on viral replication.

Requirement of Env immunosuppressive function for viral spread in vivo. To assess the role of Env-induced immunosuppression during retroviral infection, the Inventors compared the in vivo propagation of WT and DM F-MLV by quantifying viral loads in mouse serum. This work was carried out using Swiss mice, which were selected because (i) they are highly sensitive to infection with the N-tropic F-MLV and (ii) as non-congenic outbred animals they constitute a more "real life" model host than any inbred mouse strain. In this model, as illustrated in Figure 2A, WT F-MLV first developed a high viremia in all animals during the primary infection phase (peak at day 7 after virus injection), followed by the establishment of a persistent infection with viremia reaching a plateau. In contrast, the non-immunosuppressive DM F-MLV was absent (or barely detectable in 4 out of 10 mice) at day 7, and undetectable 14 days after injection (as similarly observed when using even higher doses of virus, see Figure 4). Importantly, mice immunocompromised by 5 -Gray X-ray irradiation prior to infection displayed similar viral loads when injected with either WT or DM F-MLV, at least during the first 2 weeks of infection (Figure 2B), thus excluding that DM F-MLV is deficient in any step of its in vivo replication, consistent with the in vitro data (Figure IB and Figure ID). However, DM F-MLV was eliminated when the mouse immune system recovered from

irradiation, with no more viremia detected 4 weeks post-infection. These experiments thus demonstrate that retroviral Env is not solely a "mechanical" component of the viral particle driving entry into target cells, but also a critical effector in the host-parasite relationships, essential for MLV infection.

Role of NK cells in virus control. To understand the mechanism involved in the ISD-dependent viral escape from the host immune system, the Inventors searched for immune effectors responsible for rejection of the mutant F-MLV, reasoning that since these effectors were not able to eliminate WT F-MLV, they should be -directly or indirectly- the target of Env-driven immunosuppression. Due to the rapid "control" of DM F-MLV (Figure 2A), the Inventors suspected a role for innate immune effectors, and therefore tested the possible involvement of NK cells, known for their antiviral activity, upon in vivo cell depletion. As not all the animals from the outbred Swiss mouse strain were found to express the NKl. l antigen, a surface marker specific for NK and NKT cells in certain mouse strains, NKl .l + mice were first sorted based on their blood cell NKl .l expression, before being treated with the anti-NK1.1 antibody. As shown in Figure 3 A, NK1.1+ cell depletion enabled the propagation of DM F-MLV, with viral loads similar to those observed with WT F-MLV when assayed 5 days post-infection. As a control, this effect was not observed upon treatment of NKl . l -negative Swiss mice by the anti-NKl .l antibody, with no viremia detected for DM F-MLV (see Figure 6). Accordingly, an NK1.1+ cell subset, most probably NK cells, efficiently and rapidly eliminates DM F-MLV, but fails to block WT virus. However, propagation of DM F-MLV in the antibody-treated mice was transient and the virus was completely eliminated by 7 days after infection, despite maintenance of the NK-depleted state. This observation indicates that a second mechanism for virus clearance, independent of the NKl .l + cells, takes over at a later time to eradicate the mutant virus, with a kinetics compatible with the establishment of adaptive immunity.

Role of cytotoxic T lymphocytes in virus control. To demonstrate the role of the adaptive immune response in the second phase of mutant F-MLV elimination, the Inventors used athymic Swiss Nude mice. As above, depletion of the NKl .l + cells subset first allowed the propagation of DM F-MLV, to the same extent as WT F-MLV (Figure 3B). But this time, DM F-MLV viremia persisted as long as NK cell depletion was maintained, consistent with the virus rejection observed in normal mice in the absence of NK cells (Figure 3A) being

driven by the adaptive immune system. Incidentally, this experiment also shows that the NK1.1+ cells responsible for early rejection of DM F-MLV are NK, and not NKT cells, the latter requiring the thymus to differentiate [Benlagha K, et al. (2002) Science 296:553-5]. These results demonstrate that NK cells per se are sufficient to eradicate a non-immunosuppressive F-MLV both at the early stage of infection, and also at a later stage, when the virus is then additionally cleared by a T cell-dependent mechanism.

To characterize further the latter process and the likely involvement of CD8 T cells, the Inventors again used depleting antibodies, directed against the CD8cc cell surface marker, which led to CD8 T cell elimination (Figure 3C). In Swiss mice simultaneously depleted for NK and CD8 T cells, DM F-MLV persisted in the serum as long as the treatment lasted, whereas it was not detected at any time in mice only treated with the anti-CD8 antibodies (Figure 3C) or with a control antibody. Thus, IS activity of the WT F-MLV Env allows the virus to escape two antiviral, NK cell- and CD8 T cell-dependent, immune activities.

Of note, a similar depletion procedure applied to CD25+ cells (thus including the natural Treg cells) before F-MLV infection had no effect on WT F-MLV viremia in vivo and, as expected, did not allow DM F-MLV to propagate (see Figure 7 and Discussion).

Protective activity and immunogenicity of the mutant F-MLV. The data in Figure 2 and Figure 3 clearly show that the mutant, immunosuppression-negative F-MLV, does not propagate in immunocompetent mice, and as such behaves as a bona fide live attenuated virus. To determine whether it could act as a vaccine, a series of Swiss mice were first injected with DM F-MLV, the absence of viremia was checked 4 weeks post infection, and mice were finally challenged with WT F-MLV. As shown in Figure 4A, all the "vaccinated" mice were protected against challenge, with none of them developing significant viremia, under conditions where all mock-vaccinated (PBS) mice became viremic. Clearly, the mutant virus has generated protective immunity against WT F-MLV. Of note, attempts to detect virus-specific T cell responses by IFN-γ ELISpot were not successful. Furthermore, antibodies against F-MLV SU, TM and Gag proteins were detected in the serum of the "vaccinated" mice, but IgG levels were low (twice the background level, see Figure 8A, 8B, 8C), with no detectable neutralizing activity (Figure 8D). Clearly, as similarly observed for other retroviruses and animal models [Letvin NL, et al. (2007) J Virol 81:12368-74; Mansfield K, et al. (2008) J Virol 82:4135-48], the protective immunity induced by the attenuated F-MLV is not associated with high immune responses.

To characterize further the immune response induced by the non-immunosuppressive F-MLV, under well-defined conditions, the Inventors chose to inject UV-inactivated F-MLV in inbred C57B1/6 mice, in order to (i) work with the same amounts of antigen for WT or DM F-MLV and (ii) analyze specific T cell responses against well defined immunodominant H-2b-restricted antigenic peptides [Chen W, et al. (1996) J Virol 70:7773-82 ; Iwashiro M, et al. (1993) J Virol 67:4533-42]. As shown in Figure 4, not only humoral but also cellular virus-specific responses could be detected, although they were still found to be very low as above. Interestingly, Figure 4 also shows that injection of the non-immunosuppressive DM F-MLV reproducibly induced significantly higher responses than its wild type counterparts, for both anti-TM and anti-Gag IgG and for IFN-γ secretion by CD4 and CD8 T cells, suggesting that Env-mediated immunosuppression impairs, at least quantitatively, the induction of anti-viral responses. This inhibitory effect was still observed when recombinant proteins of only 64 aa, corresponding to the Env ectodomains, were injected : a 40-fold increase in the IgG response was detected in the case of DM versus WT F-MLV ectodomain injection (see Figures 9). A large increase was also observed for the TM ectodomains mutated along the same rules for the closely related, recently identified human XMRV retrovirus [Urisman A, et al. (2006) PLoS Pathog 2:e25], as well as for the human HTLV retrovirus (Figure 9). These results indicate that mutation of the retroviral ISD could be useful to improve viral antigen immunogenicity for future vaccine strategies.

DISCUSSION

Thus far, the physiological role of retroviral Env immunosuppressive function remained elusive. In the current study, the Inventors succeeded in "switching off the immunosuppressive function of F-MLV Env without altering its fusogenic activity, by replacing 2 specific aa in its TM subunit according to the rule that the Inventors had previously established for other retroviral Envs [Mangeney M, et al. (2007) Proc Natl Acad Sci U S A 104:20534-9]. While the ISD overlaps the very constrained helix- loop-helix segment of the Env TM ectodomain that is believed to play a pivotal role in its conformational changes during the fusion process, this double mutation preserved its "mechanical" fusogenic function in a series of in vitro infectivity assays, including infectivity of pseudotypes, and replication efficiency of reconstructed mutant F-MLV proviruses. Indeed, the non-immunosuppressive DM F-MLV Env could be introduced back into the complete F-MLV cloned provirus, resulting in a mutant F-MLV retrovirus that disclosed infectivity and replication efficiency in vitro identical to that of WT virus, as assayed on NIH/3T3 cells with either the "basic" virus or an IRES-g p-marked version of the virus. Full conservation of the functionality of DM F-MLV was further ascertained by the in vivo infection assay performed on immunocompromised X-ray- irradiated mice, which disclosed viremia identical to that of the wild-type virus, at least in the initial stages of infection. Thanks to the achieved genetic disjunction between the Env fusogenic and immunosuppressive activities, the physiological impact of the IS function carried by a retroviral Env could then be unambiguously and specifically determined. Remarkably, the Inventors could demonstrate that the non-immunosuppressive DM F-MLV is unable to propagate in untreated, immunocompetent mice, under conditions where such mice become persistently infected by WT F-MLV. Again, lack of viral propagation of DM F-MLV in vivo is not dependent on some "mechanical" defect of the virus but actually depends on loss of the Env-associated IS function, viremia being undistinguishable from that of WT virus in the X-irradiated mice assayed in parallel. These conclusions are further strengthened by the specific NK and/or CD8 T cell depletion experiments that were carried out with DM virus, which resulted in recovery of viral loads as high as those observed with WT virus at varying stages of infection, consistent again with full intrinsic replicative "competence" of the mutant virus but involvement of a viral-associated "immune" activity restricted to the wild-type virus. Clearly, retroviral Envs are not solely a "mechanical component" of the viral particle driving entry into the target cell, but also a critical immunological, "cytokinal" effector in the host-parasite relationships, absolutely required for viral spread.

Another important outcome of the study is the identification of a dual immune activity of the viral ISD. Indeed, the Inventors show that when F-MLV is deprived of its IS function, NK cells alone can efficiently prevent primary infection (Figure 3B, 3C), and even decrease viral loads to undetectable levels in case of an established viremia (Figure 3B), whereas they fail to do so with the wild-type virus. On the other hand, CD8 T cells alone also prove to be sufficient to eliminate the mutant virus, even when the viral load has reached high levels (Figure 3A), whereas again they fail to do so with the wild-type virus. Altogether, these experiments show that both the innate -NK cells- and the adaptive -CD8 T cells- immune systems are able to prevent in vivo spread as well as persistence of the non immunosuppressive mutant F-MLV, and that they are both -directly or indirectly- inhibited by the ISD of the wild-type immunosuppression-positive F-MLV.

The precise mechanism by which the now clearly identified retroviral IS function inhibits the innate and adaptive antiviral immune response at the molecular level is yet largely unknown, but a series of putative cellular mediators can be inferred. Among them, Treg cells are known to suppress both CD8 T and NK cell functions [Rouse BT, et al. (2006) Immunol Rev 212:272-86 ; Ralainirina N, et al. (2007) J Leukoc Biol 81:144-53] and have been implicated by several authors in retroviral invasion and persistence [Schneider-Schaulies S, Dittmer U (2006) J Gen Virol 87: 1423-38; Robertson SJ, et al. (2006) J Immunol 176:3342-9]. Several studies have shown impairment of virus-specific CD8 T cell responses by Treg cells in chronic infection by the F-MLV/SFFV complex [Dittmer U, et al.. (2004) Immunity 20:293-303; Iwashiro M, et al. (2001) Proc Natl Acad Sci USA 98:9226-30; Myers L, et al. (2009) J Immunol 183: 1636-43 ; Zelinskyy G, et al. (2005) J Virol 79:10619-26 ; Zelinskyy G, et al. (2006) Eur J Immunol 36:2658-70; Zelinskyy G, et al. (2009) Blood 114:3199-207], which could therefore be activated and/or induced by the F-MLV Env-associated IS activity. However, the cell depletion experiments that the Inventors carried out to eliminate CD25+ natural Treg cells during the early phase of F-MLV infection had no effect on viremia, excluding a pivotal role of these cells. Other studies, using the F-MLV/SFFV complex, have also shown that Treg cell inhibition or depletion is not sufficient for virus elimination, [Zelinskyy G, et al. (2006) Eur J Immunol 36:2658-70; Zelinskyy G, et al. (2009) Blood 114:3199-207; He H, et al. (2004) J Virol. 78:11641-7]. Thus, Treg cells -either natural or induced- are most probably not primary effectors of Env-mediated immunosuppression. Dendritic cells (DC) are also known to be essential for both NK cell activation and CD 8 T cell priming [Andrews DM, et al. (2005) Mol Immunol 42:547-55; Guermonprez P, et al. (2002) Annu Rev Immunol 20:621-67], and are good candidate for being involved in the control of F-MLV in vivo. Further work will be required to determine whether DC functions (e.g., migration, cytokine secretion) are altered and -possibly- differentially affected by F-MLV in its wild-type and immunosuppression-negative versions.

Whatever the mechanism underlying Env-mediated immunosuppression, the present investigation has another important outcome, as it suggests a simple method for improving antiviral vaccines. Indeed, besides identifying a target for anti-retroviral strategies, the Inventors provide evidence that inhibition of ISD activity -through appropriate mutations-significant ly improves immunogenicity of both recombinant Envs and inactivated virions. Indeed, with the immunosuppression-negative mutants, the Inventors observed a significant increase in both the B and T cell responses against antigenic determinants carried by the viral Env and Gag proteins. Since the mutation strategy the Inventors devised minimally alters antigenicity, with only 2 aa being mutated within the whole viral proteins, and with the structure and fusion function of the mutated Env being unaltered, it is likely that the increased immunogenicity of the mutant, "optimized" antigens results from the abolition of the IS activity naturally carried along with them. If general, this finding could provide a way to dramatically improve currently unsatisfactory vaccination attempts directed against retroviruses, including those pathogenic in humans.

Along this line, the results presented for the human HTLV and XMRV retroviruses (Figures 9), disclosing a very significant increase in B cell responses following introduction of specific mutations in their identified ISD associated with the inhibition of their IS activity, should augur well for the proposed vaccine strategy.

MATERIALS AND METHODS

Mice and cells. Swiss (N-tropic F-MLV permissive), Balb/c and C57BL/6 mice, 6-10 weeks old, were from Janvier (Laval, France). Swiss-Nude mice were from Institut Gustave Roussy breeding center. 293T (CRL11268), HeLa (CCL2), NIH/3T3 (CRL-1658), and MCA205 cells [Suzuki T, et al. (1995) J. Exp. Med. 182:477-486] were cultured in DMEM with 10 % FCS, streptomycin (100 μg/mL) and penicillin (100 units/mL). Mouse splenocytes were cultured in RPMI with 10 % FCS, antibiotics as above and 5 x 10"5 M β-mercaptoethanol.

Plasmids. phCMV-envF-MLV was constructed by inserting the F-MLV env gene retrieved by PCR from p57 ([Sitbon M, et al. (1990) J Virol 64:2135-40]; gift from Dr Mougel) using primers 1 and 2 (see SI text for primer sequences) and digested with Xhol and Mlul, into phCMV-envHERV-T [Blaise S, et al. (2003) Proc Natl Acad Sci U S A 100:13013-8], digested with the same enzymes. Each mutant derivative was constructed by a three- fragment ligation of phCMV-envF-MLV opened by Clal/Avrll, and two PCR products generated with primer pairs 3-4 and 5-6 for the E14R mutation, 3-7 and 6-8 for the A20F mutation, and 2-3 and 6-8 for the E14R+A20F mutation, restricted with Clal and Avrll. The p57 DM F-MLV was constructed by inserting the BstZl lI/BsmI fragment of the phCMV-envF-MLV double mutant into p57 digested with the same enzymes. The pDFG retroviral vector expressing the WT or DM F-MLV Env (and hygromycin resistance) for stable transduction of MCA205 cells was constructed by inserting the BspEI/MluI fragment from phCMV-envF-MLV into pDFG-MoTMl (10) digested with AgEI/MluI. The bacterial expression vectors for WT and DM F-

MLV Env ectodomains were constructed by inserting a PCR fragment, generated with WT or DM phCMV-envF-MLV as a template and primers 9-10 and digested with Ncol/Xhol, into pET28(+)b (Novagen) digested with the same enzymes.

Infectivity assay with pseudotyped virions. Mo-MLV virions pseudotyped with WT or DM F-MLV Env were produced as in [Blaise S, et al. (2004) J Virol 78:1050-4] by cotransfecting 7.5 x 105 293T cells with 0.55 μg of phCMV-envF-MLV, 1.75 μg of a Mo-MLV gag-pol vector [Blaise S, et al. (2004) J Virol 78:1050-4] and 1.75 μg of a LacZ-marked defective retroviral vector (pMFGsnls/acZ), by calcium phosphate precipitation (Invitrogen). 36 hours post transfection, viral supernatants were harvested, filtered through 0.45^m-pore-size membranes, and 5-500 μΐ of supernatant supplemented with 8 μg polybrene/mL were transferred onto NIH/3T3 target cells (seeded in 24-well plates at 104 cells per well the day before infection). Viral titers were measured by X-Gal staining 3 days post infection, and expressed as lacZ ffu/mL of viral supernatant.

In vivo tumor-rejection assays. The assay was performed as in (10). Briefly, MLV virions containing the WT or DM F-MLV env-expressing pDFG retroviral vector (see Plasmids) were produced by transfecting 293T cells with 1.75 μg of the pDFG vector plus non-retroviral expression vectors for the MLV proteins (0.55 μg for MLV env and 1.75 μg for MLV gag-pol, ref. 33). Released particles were then used to transduce MCA205 cells (5 x 105 cells). Cells were cultured in selective medium (400 units/mL hygromycin) for 3 weeks, and finally scraped without trypsination to be inoculated subcutaneously into the mouse flank as in [Mangeney M, Heidmann T (1998) Proc. Natl. Acad. Sci. U. S. A. 95:14920-14925]. Tumor area (mm2) was determined by measuring perpendicular tumor diameters thrice weekly, and extent of "immunosuppression" quantified by an index based on tumor size: (Aenv-Anone)/Anone, where Aenv and Anone are the mean areas at the peak of growth of tumors from mice injected with env-expressing or control cells, respectively.

F-MLV production and assay. 7.5 x 105 293T cells were transfected with 4 μg of WT or DM p57 DNA using calcium phosphate transfection (Invitrogen). Cell supernatants were collected 48 h later, filtered through 0.45^m-pore-size membranes, supplemented with 8 μg/mL polybrene and used to infect 5 x 105 NIH/3T3 cells. Infected cells were cultured for 4 additional days, expanded, and supernatants collected from day 4 to 8 post-infection. Viral particles were concentrated by ultracentrifugation, resuspended in PBS, and frozen for further use. Viral RNA (from 2 μΐ of concentrated virus or 20 μΐ of cell supernatant or 20 μΐ of mouse serum) was extracted using the RNAeasy microkit (QIAgen), reverse-transcribed using the MoMLV reverse transcription kit (Applied Biosystems) with random hexamers as primers, and the cDNA was quantified by real-time PCR using the Platinum SYBR Green qPCR kit (Invitrogen) and primers CTCAGGGAGCAGCGGGA (SEQ ID NO : 24) and TAGCTTAAGTCTGTTCCAGGCAGTG (SEQ ID NO : 25). Only values >104 RNA copies/mL are significant.

In vitro replication of WT and DM F-MLV was assayed upon infection of 104 NIH/3T3 cells (in 6-well plates) with 107 viral copies, supernatants were collected and viral loads measured as above over a 3-day period. In vivo replication of WT and DM F-MLV was assayed upon infection of Swiss or Swiss-Nude mice by intravenous injection of 107 viral copies in PBS, and viral loads measured in blood samples (retro -orbital bleed). UV-inactivated WT and DM F-MLV virions were obtained by irradiation under the cell culture hood at 5 x 105 μ.Ι/αη2.

Immune cell depletions. PK136, YTS169 and PC61 hybridomas producing depleting antibodies against the NK1.1, CD8cc and CD25 antigens, respectively, were from Dr L. Zitvogel. Ascites were produced in Nude mice and antibodies were purified by FPLC using protein A columns. NK cell depletions were performed by intraperitoneal injection of 300 μg of anti-NKl . l antibodies at day -3, 0, 3, 7 and 10 post-infection for Swiss mice. For Nude mice, two additional injections were performed at day 13 and 17. CD8 T cell depletions were performed by injecting 100 μg of anti-CD8 antibodies at day -1, 0, 5 and 10 post-infection. CD25 depletions were performed by intraperitoneal injection of 100 μg of anti-CD25 antibodies the day before infection. Depletions were checked at day 5 post-infection by quantifying NK, CD8 and CD25 cells in mouse blood by flow cytometry, using the 20d5 anti-NKG2A/C/E (Serotec), 53-6.7 anti-CD8 (Miltenyi Biotec), and 3C7 anti-CD25 (BD Biosciences) antibodies, respectively.

Recombinant proteins, peptides and oligonucleotides. Recombinant proteins were produced as in [Mangeney M, et al. (2007) Proc Natl Acad Sci U S A 104:20534-9] using BL21 (DE3) E.coli cells (Stratagene) and pET28(+)b-derived expression vectors (see Plasmids). Recombinant WT and DM TM subunit ectodomains and Gag protein were soluble and were purified on HiTrap Chelating HP columns (Amersham). WT and DM ectodomains

were further purified through a Superdex 75 HR10/30 column (Amersham) to isolate the -major- trimeric form. The synthetic GagL85_93 (CCLCLTVFL (SEQ ID NO: 57)) peptide corresponding to the immunodominant Derestricted CD8+ T cell epitope from F-MLV Gag [Chen W, et al. (1996) J Virol 70:7773-82] and the synthetic H19-Envi22-i4i (EPLTSLTPRCNTAWNRLKL (SEQ ID NO: 58)) peptide corresponding to the immunodominant I-Ab -restricted CD4+ T cell epitope from F-MLV gp70 [Iwashiro M, et al. (1993) J Virol 67:4533-42] were from Sequentia (Evry, France). CpG oligonucleotides (ODN 1826; TCCATGACGTTCCTGACGTT (SEQ ID NO : 26), CpG ODN) and control ODN (ODN 1982; TCCAGGACTTCTCTCAGGTT (SEQ ID NO : 27)) were synthesized by Proligo (France).

Anti-F-MLV antibodies detection and IFN-γ assay. IgG levels in serially diluted mouse sera were assayed by indirect ELISA using MaxiSorp microplates (Nunc, Denmark) coated with recombinant Gag or TM subunit ectodomains (2 μg/mL), anti-mouse IgG antibodies conjugated to HRP (Amersham), and BD-Opteia revelation kit (Sigma). Mouse anti-Hiss antibody (QIAgen) was used for standardization. Splenocytes from immunized mice were stimulated in vitro with or without 10 μg mL of GagL85-93 or H19-Envi22_i4i peptide, and the supernatants harvested 72 h later. IFN-γ production was then measured by sandwich ELISA, using Maxisorp plates coated with an unconjugated anti-IFN-γ antibody (R4-6A2; BD Biosciences) and detection with a biotinylated antibody (XMG1.2; BD Biosciences). Plates were developed using streptavidin-HRP (BD Biosciences) and o-Phenylenediamine (Sigma-Aldrich), and standardized using murine recombinant IFN-γ (BD Biosciences). IFN-γ levels are given after subtracting nonspecific IFN-γ secretion in the absence of stimulating peptides.

Materials and Methods for Figures 5 to 9

Plasmids:

p57-lKES-gfp plasmids are derived from the F-MLV containing p57 plasmid (see above) by introducing an IKES-gfp sequence 3' to the F-MLV env gene. Both the WT and the immunosuppression-negative DM versions were constructed, by first introducing Sail and Mlul restriction sites 3' to the env gene (via a three-fragment ligation between WT or DM p57 opened by Xhol and Bsml, and two PCR products generated with primer pairs S1-S2 and S3-S4). These modified p57 plasmids were then opened by Sall-Mlul, Klenow-treated and ligated with a blunt-ended PCR fragment amplified from p-MIGRl [Bowie AG, Unterholzner

L (2008) Nat Rev Immunol 8:911-22] using primer pair S5-S6 to introduce the IKES-gfp unit. These plasmids were then used as in Figure 1 to generate the WT and DM GFP-marked F-MLV virions.

The pET28-based and pDFG-based expression vectors for the WT and DM ectodomains of syncytin-1 were previously described [Hansen TH, Bouvier M (2009) Nat Rev Immunol 9:503-13].

To generate pDGF expressing WT and DM F-MLV Env ectodomains, PCR amplification was performed using primer pair S7-S8 and WT and DM phCMV-envF-MLV plasmids as templates. Sfil/Mlul digested PCR fragments were then introduced into the pDFG vector opened with the same enzymes.

The XMRV Env ectodomain encoding DNA sequence was generated by ligation of 3 sets of paired 70-75 oligomers with 4 nt cohesive ends for each oligomer, including a Sfil and Mlul restriction site for the external fragments. The ligation product was introduced into the Sfil/Mlul opened pDFG vector. This plasmid was used as a template for PCR amplification of the XMRV Env ectodomain with primer pair SI 1-S12 to introduce Ncol and Xhol restriction sites and the PCR fragment was introduced into the Ncol/Xhol digested pET28 plasmid. Mutations were then introduced into this plasmid using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) to generate the DM pET28 counterpart using primer pair SI 3-S14. The DM XMRV Env ectodomain was introduced back into pDFG after PCR amplification with primer pair S9-S 10.

pCMV-HTLVenv was a gift from C. Pique. The DM pCMV-HTLVenv mutant was generated by triple ligation of PCR fragments generated with primer pairs S15-S16 and S17-S18, digested with Kpnl and Nsil respectively, and the opened Kpnl-Nsil restricted pCMV-HTLVenv. The WT and DM HTLV Env ectodomains were then PCR-amplified with primer pair S19-S20, digested with Sfi-Mlu, and introduced into pDFG opened with the same enzymes. pMal bacterial expression vectors for fusion proteins with E. coli MBP were constructed by a three- fragment ligation of pMal-c2x opened with Bglll and Hindlll, a Bglll and Pstl cut PCR fragment generated with primer pair S21-22 using pMal-c2x as a template, and a Pstl and Hindlll cut PCR fragment generated with primer pair S23-24 using pCMV-HTLVenv as a template. These vectors encode a 84-residues long HTLV ectodomain fused to the C-terminus of MBP through a tri-alanine linker, identical to the fusion protein that has been crystallized [Jonjic S, et al. (2008) Curr Opin Immunol 20:30-8]. The empty pMal-c2x vector encodes the 85-residues long alpha subunit of E. coli β-galactosidase fused to the C- terminus of MBP, and was used as a control protein. MBP fusion proteins were produced as indicated in main text "Materials and Methods", and purified on cross-linked amylose resin (New England Biolabs) with PBS as a binding and washing buffer, and 20 mM Tris-Cl, 5 mM maltose pH=7.5 as an elution buffer. A second purification step on a MonoQ 5/50 GL column (Amersham) in 20 mM Tris-Cl pH=7.5 with a 0 - 1 M NaCl linear gradient was performed to separate the fusion proteins from endogenous E. coli MBP. The fusion proteins were ultimately purified on a Superdex 200 HR 10/30 column (Amersham) to isolate the trimeric form.

Primer list:

N° SEQ ID SEQUENCE

SEQ ID

51 AGGATTGTTTAACAGATCCCCCT

NO :28

SEQ ID

52 N0 : GCGACGCGTATGTATGTCGACTTATCATGGCTCGTATTCTAGTGGTTTTA 29

SEQ ID

53 N0 : ATACATACGCGTATAAAAGATTTTATTTAGTTTCCAGAAAAAGG

30

54 SEQ ID CGCGGCTACAATTAATACATAACCTTA

NO :31

s5 SEQ ID ATACATTTAATTAACTCTCGAGGTTAACGAATTCCG

NO :32

s6 SEQ ID ATACATACGCGTCTTACTTGTACAGCTCGTCCATGC

NO :33

s7 SEQ ID ACATGGCCCAGCCGGCCATGGCTGCCGTACAAGATGATCTC

NO :34

s8 SEQ ID GTATACGCGTTTATACTAGGCCTGTATGGTCAGC

NO :35

S9 SEQ ID ACATGGCCCAGCCGGCCCTCCAGGCAGCCATAC

NO :36

sl0 SEQ ID GTATACGCGTTTATACGCCAGTGTGGTCC

NO :37

sll SEQ ID ATACATCCATGGGGCTCCAGGCAGCCATAC

NO :38

sl2 SEQ ID ATGTATCTCGAGATCTCTTACTACGCCAGTG

NO :39

sl3 SEQ ID CTGTTCCTAAAAAGAGGAGGATTATGTGCTTTCCTAAAAGAAGAATGC NO : 40

sl4 SEQ ID GCATTCTTCTTTTAGGAAAGCACATAATCCTCCTCTTTTTAGGAACAG NO : 41

sl5 SEQ ID CCCCTTTTCCTTGTCACCTGTTCC

N° SEQ ID SEQUENCE

S16 SEQ ID TCCTCCTCGCTCCCAGAACAGGAGATCAAGGCCTCGTCTG

NO : 43

S17 SEQ ID CGAGGGGAGGTGTCGTAGCTGA

NO : 44

S18 SEQ ID TTATGCAAATTTTTACAAGAACAGTGCTGTTTTCTGA

NO : 45

S19 SEQ ID ACATGGCCCAGCCGGCCCTCCTACATGAGGTG

NO : 46

S20 SEQ ID GTATACGCGTTTAATGGGAATTGGTAATATTC

NO : 47

S21 SEQ ID CTGAAATCACCCCGGACAAA

NO : 48

S22 SEQ ID ATACATGGCTGCAGCATTAGTCTGCGCGTCTTTCAGG

NO : 49

S23 SEQ ID ATACATGCTGCAGCCATGTCCCTCGCCTCAGGAA

NO : 50

S24 SEQ ID ATACATAAGCTTTTAATTCTCAAGGGGGGGTCTTTC

NO : 51

Detection of neutralizing antibodies:

Neutralizing antibodies were tested by their ability to inhibit infection of NIH/3T3 target cells by F-MLV, as described for another retrovirus in Langhammer et al. [Malim MH, Emerman M (2008) Cell Host Microbe 3:388-98] . 50 of F-MLV (2 x 106 copies/mL) were incubated with serial dilutions of heat-inactivated serum for 45 min at 37°C and then transferred to NIH/3T3 cells seeded at 5000 cells per well into 96-well microplates. Three days postinfection, DNA was extracted (3 freeze-thaw cycles and proteinase K digestion), and F-MLV integrated pro viruses were quantified by real-time PCR using the 18S rRNA gene as a reference control (using the Applied BioSystems primer set).

REFERENCES

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