EP2756087A2

EP2756087A2 - Non integrative chimeric lentiviral genomes as vaccines against hiv-1

Info

Publication number: EP2756087A2
Application number: EP12756752.7A
Authority: EP
Inventors: Yahia Chebloune; Delphine ALDEBERT; Géraldine ARRODE-BRUSES
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Grenoble Alpes; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Priority date: 2011-09-12
Filing date: 2012-09-12
Publication date: 2014-07-23
Also published as: WO2013037841A3; JP2014527812A; IL231494A0; US9879230B2; CN103946385B; MX2014002953A; FR2979919A1; JP6324892B2; WO2013037841A2; ZA201402676B; CN103946385A; FR2979919B1; MX354571B; CA2848484C; IL231494B; CA2848484A1; US20140370051A1

Abstract

The present invention relates to nucleic acids containing chimeric non-integrating retrovirus genomes including the long terminal repeat (LTR) sequences 5' and 3' of the caprine lentivirus, the caprine arthritis/encephalitis virus (CAEV), or another retrovirus that does not integrate into human cells, and at least one viral gene of another retrovirus. The invention also relates to a vector containing such a nucleic acid, to an immunogenic or vaccine composition containing said vector or said nucleic acid, and to the use thereof for treating and/or preventing an infection by a retrovirus or disease induced by a pathogen.

Description

Non integrative chimeric lentiviral genomes as innovative vaccines against HIV-1

The subject of the present invention is nucleic acids comprising non-integrative chimeric retroviral genomes comprising the 5 'and 3' long terminal repeat (LTP) sequences of the goat lentivirus: arthritis-encephalitis virus Caprine (VAEC, or CAEV for Caprine Arthritis Encephalitis Virus in English) or another retrovirus not integrating into human cells and at least one viral gene of another retrovirus. The invention also relates to a vector comprising such a nucleic acid, an immunogenic or vaccine composition comprising said vector or nucleic acid, and their use for the treatment and / or prevention of infection by a retrovirus or a disease induced by a pathogen. At present, the development of effective vaccines against retroviral infections is a major public health issue at the global level. Recently, vaccines based on the use of vectors having the ability to express immunogenic proteins in the vaccinated host have been developed. These vaccine vectors, after amplification in bacteria, are purified and directly injected into the host requiring vaccination. The vector is thus supported by the host cells, the immunogenic proteins are expressed and presented to the molecules of the major histocompatibility complex of class I and II, thus making it possible to generate immune responses against these immunogenic proteins. The first vaccination tests using vector retroviral vaccines made it possible to show that immunization against Rous sarcoma virus (Chebloune et al., 1991, J. Virol, 65, 5374-5380), the virus of the New Castle disease (Cosset, Bouquet et al., 1991, Virology 185, 862-866) and then influenza (Robinson, Hunt and Webster, 1993, Vaccine, 11 (9): 957-960) was possible in the chicken.

This vaccine approach is particularly interesting for the fight against the human immunodeficiency virus (HIV). Acquired Immunodeficiency Syndrome (AIDS) continues to be a global public health problem today with more than 33 million people infected, and with more than 2 million deaths and nearly 3 million new HIV infections. year. Africa is the most affected continent, but the infection is growing rapidly in Asia and some Eastern European countries, probably due to lack of means for early detection and lack of treatment. 'infection. In addition, due to economic limitations, many HIV-infected patients in developing countries receive no treatment, and thus contribute to the massive spread of the infection. The economic impact of AIDS will certainly be very important in the coming years. In Europe, AIDS remains one of the most important communicable diseases, with around 1 million people living with AIDS and more than 20,000 new infections a year in Western and Central Europe; and with approximately 1.5 million people living with AIDS and more than 200 000 new infections per year in Eastern Europe. The development of a prophylactic vaccine that stops this infection remains a priority.

Despite many efforts, there is currently no safe and satisfactory vaccine for the protection of humans against HIV infection or the pathogenesis of HIV. Nevertheless, the many studies carried out have accumulated valuable knowledge to understand the failures of vaccine strategies used so far, and to define the required properties of a vaccine inducing protective immune responses against lentiviruses responsible for AIDS.

In particular, the vaccine must induce a CD8 + T lymphocyte response, which is associated with the control of the virus during primary infection, and the presence of which has been shown to be essential for the control of viral load in infected non-human primates ( Jin et al., 1999, J Exp Med, 189: 991-998). In addition, cytotoxic T lymphocytes (CTLs) are present in long-term non-progressing patients (LTNP) (Rinaldo et al., 1995, J Virol, 69: 5838-5842), or in exposed but non-susceptible subjects. infected with HIV (Makedonas et al., 2002, AIDS, 16: 1595-1602). These and other elements show the importance of such responses in the control of viral replication and / or disease prevention.

In addition, the vaccination must induce a CD4 + T cell response, which is essential for the stimulation and maintenance of the anti-HIV CD8 + T cell response (Kalams et al., 1999, J Virol, 73: 6715- 6720). CD4 + T cells are also essential for setting up and maintaining the antibody-based response produced by B-lymphocytes (LBs). Thus, it has been shown that SC-infected and LB-depleted macaques control their viral load less well than control monkeys (Johnson et al., 2003, J Virol, 77: 375-381). In view of these results and precedents, it therefore seems necessary that an HIV vaccine should stimulate the B and T responses of the immune system.

Among the many vaccine strategies tested are those involving so-called lentiviruses attenuated. It has thus been shown that these allow to reproducibly induce the best protection against homologous and heterologous challenge viruses (Yankee et al., 2009, Virology, 383: 103-1, Genesca, McChesney and Miller, 2009, J Intern Med, 265: 67-77 Reynolds et al., 2008, J Exp Med, 205: 2537-2550, Amara et al., 2005, J Virol, 79: 15356-15367, Whitney and Ruprecht, 2004, Curr Opin Infect Dis, 17: 17-26). However, because of their irreversible integration into the host genome and the likelihood of recurrent infection associated with proviral latencies, these viruses are pathogenic in some adults and in newborns (Desrosiers, 1994, AIDS Res Hum Retroviruses, 10: 331-332, Hofmann-Lehmann et al., 2003, AIDS, 17: 157-166, Baba et al., 1999, Nat Med, 5: 194-203, Baba et al., 1995, Science, 267: 1820-1825, Yankee et al., 2009, Virol, 383: 103-111). For reasons of ethics and safety, these attenuated lentiviruses can therefore never be used in the state in humans.

DNA vaccination based on viral vectors has never been associated with a development of pathologies, neither in humans nor in animals, and therefore is safer. However, tested in monkeys, these vectors have been shown to be unable to protect animals from experimental infection (Liu et al., 2006, Virology, 351: 444-454, Singh et al., 2005, J Virol, 79: 3419-3428).

There is therefore a need for new vaccine vectors for expressing lentiviral antigens at higher levels in both quantity and quality, with the aim of inducing protective responses against pathogenic viruses.

The inventors have previously described infectious viral genomes comprising a complete viral genome including the LTRs of CAEV as well as one or two genes of another retroviral genome (Bouzar et al., 2007, Virology, 364 (2): 269-280 Bouzar et al., 2004, Virology, 326 (1): 47-56, Bouzar et al., 2003, Virology, 309 (1): 41-52, Yuhai et al., 2009, Retrovirology, 6 (2). : 22). These genomes have been used to study the mechanisms of pathogenesis induced by highly pathogenic retroviruses in humans and monkeys.

The inventors have discovered that the use of the Long Term Repeat (STR or LTR) of the Caprine Arthritis Encephalitis Virus (VAEC), or CAEV, has the potential to improve the expression of vaccinating retroviral genomes and the induction of protective responses against pathogenic retroviruses, while avoiding their integration into the host cells. In particular, the inventors have demonstrated that the Caprine Arthritis Encephalitis Virus (CAEV) Repeated Terminal Sequences (LTRs) allow the constitutive expression of the genes associated with them and are not dependent on the CAEV viral tat gene. particularly the CAEV viral tat protein, to express the genes of a viral genome to which they are fused, thus allowing strong expression of viral antigens. The inventors have thus developed chimeric genomes, between the lentiviruses of SIV and HIV primates (or HIV in English for Human Immunodeficiency Virus) and CAEV, which have the properties of being non-integrative and non-replicative, while being capable of to perform a replication cycle to express all the HIV and SIV antigens present in the genomes. The inventors have demonstrated that the transfection of these genomes into primate cells (HEK293) allows the expression of all the proteins of the genes present and that these proteins are assembled into viral particles capable of carrying out a single cycle of infection (ie a pseudo-cycle) in target cells, without integration of the viral genome into these target cells. Immunization of humanized NOD / SCID mice with human mononuclear cells demonstrated the presence of strong specific humoral and cellular immune responses against viral antigens.

Definitions

"Nucleic acid" means the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine, "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine, "DNA molecules") in single-stranded form or in the form of a double-stranded helix. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid, and in particular DNA or RNA molecule, refers only to the primary or secondary structure of the molecule, and is not limited to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), viruses, plasmids and chromosomes. When referring to the structure of particular double-stranded DNA molecules, the sequences can be described herein in accordance with the normal convention which only gives the sequence in the 5 'to 3' direction along the non-transcribed strand of DNA (That is, the strand having a sequence homologous to the mRNA).

In the context of the invention, "a nucleic acid comprising a non-integrative chimeric retroviral genome" refers to a nucleic acid which comprises the cis-acting nucleic acid sequences of at least two retroviruses, said nucleic acid not being capable of to integrate into the genome of a host cell. The nucleic acid includes the 5 'and 3' Repeat Terminal Sequences (LTRs) of a first retrovirus, and at least one viral gene of a second retrovirus. By "retrovirus" is meant a virus whose genome consists of an RNA molecule and which comprises a reverse transcriptase, Le. a member of the family Retroviridae. Retroviruses are divided into three genera: oncovirus, lentivirus and spumavirus. Oncoviruses are composed in particular of the following species: murine leukemia virus (MLV), avian leucosis virus (ALV), Rous sarcoma virus (RSR, or RSV in English for Rous Sarcoma Virus), or the simian Mason-Pfizer virus. Lentiviruses are composed of the following species: Human Immunodeficiency Virus Type 1 (HIV-1), Human Immunodeficiency Virus Type 2 (HIV-1) 2, or HIV-2 for Human Immunodeficiency Virus type 2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) for Feline Immunodeficiency Virus), the Bovine Immunodeficiency Virus (VIB), the Maedi Visna virus (VMV) of sheep, the Caprine Arthritis Encephalitis Virus (CAEV) or equine infectious anemia virus (VAIE, or EIAV in English for Equine Infectious Anemia Virus). The spumavirus can be HFV. When the retrovirus is HIV-1, it can be of any serogroup, for example serogroup M (serotype AD, FH, J, K), O, N or P. When the retrovirus is HIV-2, it can be of any serogroup, for example serogroup A or B.

By "viral gene" is meant a gene present in a retroviral genome. In the context of the invention, the viral gene can be the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu or env gene.

The "gag" gene for "group specifies antigen" encodes the Gag precursor polyprotein that is cleaved to provide the basic structural proteins of retroviruses, which are capsid proteins, nucleocapsid proteins, and matrix proteins. For example, the HIV Gag protein is the precursor of the p24 capsid protein, the p6 and p7 nucleocapsid proteins, and the p17 matrix protein. By way of non-limiting example, the gag gene is the gag gene of HIV-1 (NCBI gene ID Ν 55030, updated August 20, 201 1), HIV-2 (NCBI gene ID No. 14900001, updated Aug. 27, 201 1), SIV (NCBI gene ID No. 956108, updated August 27, 201 1) or FIV (NCBI gene ID No. 1489988, updated August 20, 201 1).

The "pol" gene codes for reverse transcriptase, integrase and protease. By way of non-limiting example, the pol gene is the pol gene of HIV-1 (NCBI gene ID No. 155348, updated 27 August 201 1), HIV-2 (NCBI gene ID No. 1490001, implemented update 27 August 201 1), FIV (NCBI gene ID No. 1489989, update 27 August 201 1) or SIV (NCBI gene ID No. 956107, updated August 20, 201 1). The "viral" or "viral infectivity factor" gene encodes a protein required for the production of infectious virions. By way of non-limiting example, the bright gene is the live gene of HIV-1 (NCBI gene ID No. 155459, updated on August 7, 2011), HIV-2 (NCBI gene ID No. 1724712, updated January 21, 2010), FIV (NCBI gene ID No. 1724709, updated February 7, 2010) or SIV (NCBI gene ID Ν 490005, updated January 21

2010).

The "vpr" gene codes for the viral R protein, which plays an important role in the G2 phase cell cycle arrest and in the regulation of the transport of the cytoplasmic pre-integration complex to the nucleus, the viral replication. By way of non-limiting example, the vpr gene is the vpr gene of HIV-1 (NCBI gene ID No. 155807, updated August 7, 2011), HIV-2 (NCBI gene ID Ν 724718, updated January 21, 2010) or SIV (NCBI gene ID No. 956112, updated January 15, 2011).

The "vpx" gene codes for the viral protein X and is related to the vpr gene. By way of non-limiting example, the vpx gene is the gene vpxdu HIV-2 (NCBI gene ID No. 1724714, updated March 19, 2011) or SIV (NCBI gene ID Ν 490006, updated July 16 2011).

The "nef" gene encodes myristylated 27- to 25-kDa protein, called the Negative Regulatory Factor, which plays a key role in the depletion of CD4 cells in vivo. By way of non-limiting example, the nef gene is the nef gene of HIV-1 (NCBI gene ID 56110, updated 7 August 2011), HIV-2 (NCBI gene ID No. 1724715, updated 19 March 2011) or SIV (NCBI gene ID No. 1490008, updated July 2, 2011).

The tat gene encodes an 86- to 101-amino acid protein called Trans-Activator of Transcription that increases the transcription rate of the retroviral genome. By way of nonlimiting example, the gene / ai is the tat gene of HIV-1 (NCBI gene ID No. 155871, updated August 20, 2011), HIV-2 (NCBI gene ID Ν 724713, updated February 7, 2010) or SIV (NCBI gene ID No. 956113, updated February 7, 2010).

The gene "rev" encodes a protein called "Regulator of Virion Expression" that allows the export of viral RNA from the nucleus to the cytoplasm. By way of non-limiting example, the rev gene is the rev gene of HIV-1 (NCBI gene ID No. 155908, updated on August 7, 2011), HIV-2 (NCBI gene ID No. 1724716, updated day of May 21st

2011) or SIV (NCBI gene ID No. 1490003, updated 15 January 2011).

The "vpu" gene encodes a protein called "Viral Protein U" that is involved in viral budding and enhancement of virion release. By way of non-limiting example, the vpu gene is the vpu gene of HIV-1 (NCBI gene ID No. 155945, updated on August 7, 2011) or SIV (NCBI gene ID No. 2828723, updated from January 21, 2010). The "env" gene encodes the gp160 precursor protein which is processed and cleaved to give the gp120 and gp41 envelope proteins. By way of non-limiting example, the gene eni / is the env gene of HIV-1 (NCBI gene ID No. 155971, updated on August 7, 201 1), HIV-2 (NCBI gene ID Ν 724717). June 18, 201 1), FIV (NCBI gene ID Ν 489987, updated June 18, 201 1) or SIV (NCBI gene ID No. 1490007, updated June 18, 201 1).

For the purposes of the present application, the term "comprises" or "comprising" designates in a particular manner "consists of" or "consisting of". Nucleic acid

The inventors have demonstrated that the Caprine Arthritis Encephalitis Virus (CAEV) Repeated Terminal Sequences (LTRs) allow the constitutive expression of the genes associated with them and are not dependent on the viral iai gene to express the genes of the caprine arthritis-encephalitis virus (CAEV). a viral genome to which they are fused, thus allowing strong expression of viral antigens. In addition, the inventors have shown that the mere presence of these LTRs prevents the integration of a heterologous retroviral genome to which they are fused.

The invention thus relates to a nucleic acid comprising a non-integrative chimeric retroviral genome, wherein said chimeric retroviral genome comprises:

the 5 'and 3' Repeat Terminal Sequences (LTR) of a first retrovirus, said first retrovirus being a lentivirus, such as the Arthritis-Encephalitis Caprine Virus (CAEV), the Maedi Visna Virus (VMV) mutton, equine infectious anemia virus (EIAV), or an oncovirus or spumavirus, and

at least one viral gene of a second retrovirus, said second retrovirus not being the first retrovirus.

The LTR sequences used are preferably derived from a retrovirus that does not integrate into the genome of a "host" or "patient", said host or patient being a host or patient in the genome of which the second and / or third retroviruses can integrate. For example, when the first retrovirus is CAEV, said host or patient is not a goat but may be a man, a monkey, a cat, or a horse, or when the first retrovirus is the EIAV, said host or patient is not a horse but can be a man, a monkey, a cat, or a sheep, or when the first retrovirus is the VMV, said host or patient is not a sheep but can be a man, a monkey, a cat, or a horse.

In a particularly preferred embodiment, the "first retrovirus" is the Caprine Arthritis Encephalitis Virus or CAEV. CAEV is a type retrovirus Goat lentivirus similar to the human immunodeficiency virus (HIV), but does not cause AIDS-like pathology in its host.

"Repeated Terminal Sequences" (LTR) denotes a sequence allowing the control of transcription, that is to say comprising an enhancer, a promoter, one or more transcription initiation signal (s). , one or more transcription termination signal (s), one or more polyadenylation signal (s). In the context of the invention, the CAEV LTRs comprise an enhancer, a promoter and a transcription initiation signal as well as a transcription termination signal and a polyadenylation signal. Preferably, the 5 'and 3' LTRs of CAEV are identical and comprise or consist of the sequence SEQ ID NO: 3.

In other embodiments, Maedi Visna Virus (VMV) LTRs or EIAV Equine Lentivirus LTRs are used. The VMV 5 'LTR comprises or consists of the sequence in position 1 to 161 of NCBI Reference Sequence NC_001452.1 (updated December 8, 2008). The VMV 3 'LTR comprises or consists of the sequence at position 9106 through 9202 of the NCBI Reference Sequence NC_001452.1 (updated December 8, 2008). The 5 'LTR of the ElAV comprises or consists of the sequence in position 61 to 381 of the NCBI NC_001450 Sequence of Sequence (updated on 1 March 2010). The 3 'LTR of the ElAV comprises or consists of the sequence in position 7269 to 8289 of the NCBI NC_001450 Sequence of Sequence (updated on 1 March 2010).

In yet other embodiments, the LTRs of an oncovirus, such as murine leukemia virus (MLV), avian leukemia virus (ALV), Rous sarcoma virus (RSV), or simian Mason-Pfizer virus, or the LTRs of a spumavirus, such as HFV are used. The 5 'LTR of the MLV comprise or consist of the sequence at position 1 to 210 of the NCBI Reference Sequence NC_001702.1 (updated February 5, 201 1). The MLV 3 'LTR comprises or consists of the sequence at position 5735 to 8135 of the NCBI Reference Sequence NC_001702.1 (updated February 5, 201 1). The 5 'LTR of the ALV consists of or consists of the sequence at position 1 to 594 of the NCBI NC_0151 Reference Sequence 16.1 (updated April 18, 201 1). The 3 'LTR of the ALV consists of or consists of the sequence at position 5338 to 7489 of the NCBI NC_0151 Reference Sequence 16.1 (updated April 18, 201 1). The RSV 5 'LTR consists of or consists of the sequence in position 22 to 102 of NCBI Reference Sequence NC_001407.1 (updated December 8, 2008). The RSV 3 'LTR comprises or consists of the sequence at position 9058 to 9292 of the NCB Sequence NC_001407.1 (updated December 8, 2008). The simian Mason-Pfizer virus 5 'LTR comprises or consists of the sequence at position 26 to 123 of the NCBI Reference Sequence NC_001550.1 (updated December 8, 2008). The simian Mason-Pfizer virus 3 'LTR comprises or consists of the sequence at position 7573 to 781 1 of NCBI Reference Sequence NC_001550.1 (updated December 8, 2008). The 5 'LTR of the HFV consists of or consists of the sequence at position 1 to 1760 of the NCBI Reference Sequence NC_001364.1 (updated 22 April 2009). The 3 'LTR of the HFV consists of or consists of the sequence at position 1 1487 to 13246 of the NCBI Reference Sequence NC_001364.1 (updated 22 April 2009).

The inventors have shown that the Caprine Arthritis Encephalitis Virus (CAEV) Repeated Terminal Sequences (LTRs) were not dependent on the viral iai gene to express the genes of a viral genome to which they are fused, advantageously the The nucleic acid according to the invention does not contain the tat gene of said first retrovirus.

In the context of the invention, the "second retrovirus" is different from the first retrovirus. It can be an oncovirus, a lentivirus or a spumavirus. For example, when CAEV LTRs are used, the second retrovirus is not CAEV. Preferably, the second retrovirus is an oncovirus, such as murine leukemia virus (MLV), avian leukosis virus (ALV), Rous sarcoma virus (RSV), or simian Mason-Pfizer virus. , a lentivirus, such as the human immunodeficiency virus type 1 (HIV-1), the human immunodeficiency virus type 2 (HIV-2), the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV) or equine infectious anemia virus (EIAV), or a spumavirus, such as HFV. Particularly preferably, the second retrovirus is HIV-1, HIV-2, SIV or IVF.

Preferably, said at least one viral gene of said second retrovirus is selected from gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes. In a particular aspect, said chimeric retroviral genome comprises at least two, three (e.g., gag, pol, vif, or gag, pol, env genes), four, five, six, seven, eight, nine, ten genes. viral of said second retrovirus. Advantageously, said chimeric retroviral genome comprises the iai gene of said second retrovirus. Even more preferably, said chimeric retroviral genome comprises the set of gag, pol, vif, vpx, vpr, nef, tat, rev, and vpu genes of said second retrovirus. In a particularly preferred aspect, said chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and S IV, HIV-1, HIV-2 or FIV genes. In a still preferred aspect, said chimeric retroviral genome comprises or consists of the sequence of the SIV retroviral genome (SEQ ID NO: 4), the retroviral genome of HIV-1 (SEQ ID NO: 2), the retroviral genome of HIV-1. 2 (SEQ ID NO: 5), or retroviral genome of FIV (SEQ ID NO: 6).

The chimeric retroviral genomes of SIV, HIV-1, HIV-2 and FIV are respectively schematically represented in FIGS. 1 to 4. In a particular embodiment, said chimeric retroviral genome further comprises at least one viral gene. a third retrovirus, said third retrovirus not being the first retrovirus, i.e., being different from said first retrovirus. Thus, for example, when CAEV LTRs are used, said third retrovirus is not CAEV. Said "third retrovirus" may be chosen from one of the retroviruses as defined above.

When said chimeric retroviral genome comprises at least one viral gene of a second retrovirus and at least one viral gene of a third retrovirus, said second retrovirus and third retrovirus are different. Said second retroviruses and third retroviruses may or may not be of different kinds, for example said second and third retroviruses may each be an oncovirus, a lentivirus, or a spumavirus, or said second and third retroviruses may be respectively (i) a lentivirus and a spumavirus, or conversely a spumavirus and a lentivirus, (ii) an oncovirus and a lentivirus, or conversely a lentivirus and an oncovirus, or (iii) a spumavirus and an oncovirus, or conversely an oncovirus and a spumavirus.

Preferably, when said chimeric retroviral genome comprises at least one viral gene of a second retrovirus and at least one viral gene of a third retrovirus, said second retroviruses and third retroviruses are each a lentivirus, and preferably, said lentivirus is selected among HIV-1, HIV-2, SIV, IVF or EIAV. Even more preferably, the second retrovirus and the third retrovirus are lentiviruses of different species, serogroup, or serotype. Thus, for example when said second retrovirus is HIV-1, said third retrovirus is HIV-2, or when said second retrovirus is serogroup M HIV-1, said third retrovirus is HIV-1 serogroup O, or when said second retrovirus is HIV-1 serogroup M and serotype 1, said third retrovirus is HIV-1 serogroup M and serotype B. Preferably, said at least one viral gene of said third retrovirus is selected from gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes. In a particular aspect, said chimeric retroviral genome further comprises at least two, three, four, five, six, seven, eight, nine or ten viral genes of said third retrovirus, preferably including the tat gene.

Even more preferably, said chimeric retroviral genome further comprises all the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said third retrovirus, that is to say that the said chimeric retroviral genome comprises the set of gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said second retrovirus and the set of gag, pol, vif, vpx, vpr, nef genes, tat, rev, vpu and env of said third retrovirus.

Said retroviral chimeric genome may therefore comprise a viral gene of said second retrovirus and nine viral genes of said third retrovirus, or two viral genes of said second retrovirus and eight viral genes of said third retrovirus, or three viral genes of said second retrovirus and seven viral genes of said third retrovirus. , or four viral genes of said second retrovirus and six viral genes of said third retrovirus, or five viral genes of said second retrovirus and five viral genes of said third retrovirus, or six viral genes of said second retrovirus and four viral genes of said third retrovirus, or seven viral genes said second retrovirus and three viral genes of said third retrovirus, or eight viral genes of said second retrovirus and two viral genes of said third retrovirus, or nine viral genes of said second retrovirus and a viral gene of said third retrovirus. By way of nonlimiting examples, said chimeric retroviral genome may therefore comprise the gag gene of said second retrovirus and the pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said third retrovirus; or the gag and pol genes of said second retrovirus and the genes vif, vpx, vpr, nef, tat, rev, vpu and env of said third retrovirus; or the gag, pol, vif genes of said second retrovirus and the vpx, vpr, nef, tat, rev, vpu and env genes of said third retrovirus; or the gag, pol, vif, vpx genes of said second retrovirus and the vpr, nef, tat, rev, vpu and env genes of said third retrovirus; or gag, pol, alive, vpx, vpr of said second retrovirus and the genes nef, tat, rev, vpu and env of said third retrovirus; or the gag, pol, vif, vpx, vpr, nef genes of said second retrovirus and the tat, rev, vpu and env genes of said third retrovirus; or the genes gag, pol, vif, vpx, vpr, nef, tat of said second retrovirus and the genes rev, vpu e \ env of said third retrovirus; or the gag, pol, vif, vpx, vpr, nef, tat, rev genes of said second retrovirus and the vpu and env genes of said third retrovirus; or the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu genes of said second retrovirus and the env gene of said third retrovirus. In a particularly preferred aspect, said chimeric retroviral genome comprises the gag, pol, vif, vpx and vpr genes of said second retrovirus and the nef, tat, rev, vpu and env genes of said third retrovirus.

In a still more preferred aspect, said chimeric retroviral genome comprises the gag, pol, vif, vpx and vpr genes of SIV and the nef, tat, rev, vpu and env genes of HIV-1, or conversely the gag, pol genes, vivid, vpx e \ vpr d HIV-1 and the genes nef, tat, rev, vpu and env of SIV. In another particularly preferred aspect, said chimeric retroviral genome comprises the gag, pol, vif, vpx and HIV-1 genes and the nef, tat, rev, vpu and env genes of HIV-2, or conversely the gag genes. , pol, alive, vpx and vpr of HIV-2 and the nef, tat, rev, vpu and env genes of HIV-1. In an even more preferred aspect, said chimeric retroviral genome comprises or consists of the sequence SEQ ID NO: 7 (a schematic representation of this chimeric retroviral genome is in FIG. 5).

In a particular embodiment, when the pol gene is present in the chimeric retroviral genome, said pol gene is a pol gene deleted sequences encoding integrase (in). Preferably, said pol gene is deleted from the sequence SEQ ID NO: 8 (SIV integrase sequence), SEQ ID NO: 9 (HIV-1 integrase sequence), SEQ ID NO: 10 ( HIV-2 integrase sequence), or SEQ ID NO: 1 1 (sequence of the FIV integrase).

Thus, in a particularly preferred embodiment, said retroviral genome comprises or consists of the sequence SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The chimeric retroviral genomes comprising or consisting of the sequence SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are respectively diagrammatically shown in Figures 6 to 10.

The invention also relates to a vector comprising a nucleic acid according to the invention.

The term "vector" refers to an extrachomosomal element by which a DNA or RNA sequence (ie, a "foreign" gene) can be introduced into a host cell, thereby transforming the host and allowing expression ( ie transcription and translation) of the introduced sequence. The extrachromosomal element can be a self-replicative sequence, a phage sequence or a nucleotide sequence, a single or double-stranded DNA or RNA, a plasmid, a cosmid. A vector typically contains the DNA of a transmissible agent, into which a foreign DNA is inserted and a selection marker. One common way of inserting a DNA fragment into another DNA segment involves the use of enzymes, called restriction enzymes, which cleave DNA at specific sites (specific nucleotide groups), called Restriction sites. Generally, the foreign DNA is inserted at one or more restriction sites of the DNA vector, and then transported by the vector into a host cell with the DNA of the transmissible agent. A segment or DNA sequence comprising an added or inserted DNA, such as a vector, may also be referred to as a "DNA construct". A common type of vector is a "plasmid", which is usually an autonomous double-stranded DNA molecule, usually of bacterial origin, that can easily accept additional (foreign) DNA and can easily be introduced into a host cell. appropriate. A large number of vectors, including plasmids, have been described for replication and / or expression in different eukaryotic and prokaryotic hosts. In the context of the invention, the vector comprises a selection marker, such as an antibiotic resistance gene, and a nucleic acid according to the invention. Preferably, the resistance gene is a resistance gene to ampicillin or kanamycin.

The nucleic acids and / or the vector according to the invention can be used to transform or transfect a host cell or organism, i.e. for the expression of the chimeric retroviral genome according to the invention.

The term "host cell" refers to any cell of any organism that is selected, modified, transformed, transfected, transduced, cultured, or used or manipulated in any way for the production of a substance by the cell, for example for the expression of a gene, a DNA sequence, a protein, a virion by the cell. In the context of the invention, the host cell is a mammalian cell. Suitable host cells include, but are not limited to, HEK293 cells, human CD4 + T lymphocyte CEMx174 and M8166, human CD4 + T cells, human CD8 + T cells, mononuclear cells of human blood.

The transformation of the host cell or organism with the nucleic acid and / or the vector according to the invention can be carried out according to the standard techniques known to those skilled in the art, for example by transfection, electroporation, microinjection, DEAE-Dextran transduction, cell fusion, calcium phosphate precipitation, or use of gene gun, or a DNA vector transporter (see for example, Wu et al., 1992, J Biol Chem 267: 963- 967, Wu et al., 1988, J Biol Chem 263: 14621-14244, Hartmut et al., Canadian Patent Application No. 2,012,311, published Mar. 15, 1990). Immunogenic or vaccine composition and uses thereof

The nucleic acid or the vector according to the invention may be used for immunogenic or vaccinal purposes.

Thus, the invention also relates to an immunogenic or vaccine composition comprising a nucleic acid or a vector according to the invention.

In the context of this application, the term "vaccine" refers to prophylactic or therapeutic vaccination.

By immunogenic or vaccine composition is meant a composition for inducing an immune response against a retrovirus as defined above. By immune response is meant a response involving T cells, for example CD4 + and CD8 + T cells, and B cells.

According to one embodiment, the immunogenic or vaccine composition according to the invention is monovalent, i.e. it allows an immune response against a single retrovirus, for example against HIV-1 or HIV-2.

According to another embodiment, the immunogenic or vaccine composition according to the invention is multivalent, ie it allows an immune response against several retroviruses, for example against HIV-1 and HIV-2 or several pathogens, for example against HIV-1 and HCV (Hepatitis C Virus). In this case, the vaccinating vector expresses the antigens of the one and the other pathogenic agent.

According to another embodiment, the immunogenic or vaccine composition according to the invention is polyvalent. Such an immunogenic or vaccine composition can be obtained by combining several immunogenic or vaccine monovalent compositions according to the invention. The immunogenic or vaccine composition may further comprise at least one other vaccine, ie an attenuated live virus, an inactivated virus or a subunit, against another virus, such as a sexually transmitted virus, such as for example hepatitis B, hepatitis C virus or papillomavirus.

In a preferred embodiment, the immunogenic or vaccine composition according to the invention comprises a pharmaceutically acceptable vehicle.

A "pharmaceutically acceptable carrier" refers to any vehicle in which the immunogenic or vaccine composition according to the invention can be formulated. This includes a saline solution such as a phosphate buffered saline. In general, a diluent or vehicle is chosen depending on the mode and route of administration, and according to standard pharmaceutical practice. A pharmaceutically acceptable carrier includes, without limitation, ion exchangers, aluminum, aluminum stearate, lecithin, self-emulsifying drug delivery systems such as D-oc-tocopherol polyethylene glycol 1000 succinate, surfactants used in dosage form pharmaceutical such as Tweens or other polymeric delivery matrices, serum proteins such as human albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, glyceride mixtures of saturated fatty acids of plants, water, salts or electrolytes, such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene polymer blocks and wool grease. Cyclodextrins such as A-, B-, and g-cyclodextrins, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2-and 3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives may also be advantageously used for to improve the delivery of the compositions according to the invention.

The compositions according to the invention may further contain an adjuvant. Any adjuvant or mixture of pharmaceutically acceptable adjuvants conventionally used in the field of vaccines can be used for this purpose. Examples of suitable adjuvants are aluminum salts such as aluminum hydroxide or aluminum phosphate and DC-Chol. Any adjuvant or mixture of pharmaceutically acceptable adjuvants conventionally used in the field of vaccines can be used for this purpose. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide or aluminum phosphate and DC-Chol.

The compositions according to the invention may contain adjuvant genes, that is to say genes that express proteins that will act as adjuvants by increasing the immunogenicity of the expressed viral proteins. For example the genes that encode cytokines such as interleukins (II) [IL-2, IL12, IL-15, ... or GM-CSF (granulocyte-macrophage colony-stimulating factor)]. These adjuvant genes are either incorporated into the vaccine plasmid or co-injected as separate expression plasmids.

Any method of administration known to those skilled in the art can be used. In particular, the nucleic acid, the vector, the immunogenic or vaccine composition according to the invention can be administered orally, by inhalation, or parenterally (in particular by intradermal, subcutaneous, intravenous, intramedullary or intramuscular injection). . When the parenteral route is used, the nucleic acid, the vector, the immunogenic or vaccine composition according to the invention may be in the form of injectable solutions and suspensions, packaged in ampoules or flasks. Forms for parenteral delivery are generally obtained in mixing the nucleic acid, the vector, the immunogenic or vaccine composition according to the invention with buffers, emulsifiers, stabilizers, preservatives, solubilizing agents. According to known techniques, these mixtures can then be sterilized and packaged in the form of intradermal, subcutaneous, intravenous, intramedullary or intramuscular injections. One skilled in the art can use buffers based on organic phosphate salts as a buffer. Examples of emulsifying agents include methylcellulose, acacia, sodium carboxymethylcellulose. Examples of stabilizers include sodium sulphite, sodium metasulfite, and examples of preservatives include sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol. The nucleic acid, the vector, the immunogenic or vaccine composition may also be in freeze-dried form.

The vaccine DNA solution can be injected directly or administered by electroporation in vivo using a commercial electroporator or encapsulated in liposomes or nanoparticles or using any in vivo transfection method that allows for better introduction of the vaccine DNA. in the cells of the vaccinated host.

In another aspect, the invention relates to a nucleic acid according to the invention, a vector according to the invention and / or an immunogenic or vaccine composition according to the invention for their use in the prevention and / or treatment of an infection. by a retrovirus.

By "prevention" or "prevention" is meant the inhibition of a retroviral infection, ie preventing the retrovirus from causing an infection, or preventing the spread of the retrovirus within an infected subject or subject to the other.

By "treating" or "treatment" is meant limiting the severity of the disease, preventing recurrent infections, i.e. limiting the reactivation of latent or persistent infections, and alleviating the symptoms of retrovirus infections. The retrovirus is as defined above, and most preferably the retrovirus is HIV-1, HIV-2 or FIV.

The term "patient" or "subject" or "host" refers to a human or non-human mammal, or a bird. Preferably, the patient is a primate, a murine (mouse), a feline (cat), a canine, an equine (a horse), a bird, a human, including women, men, adults and children.

The present invention also relates to a method of vaccination or treatment of a subject requiring it comprising the administration of a prophylactically or therapeutically effective amount of a nucleic acid according to the invention, or a vector according to the invention, or an immunogenic or vaccine composition according to the invention.

A "prophylactically or therapeutically effective amount" refers to a quantity of nucleic acid, vector or immunogenic or vaccine composition for conferring a therapeutic or prophylactic effect on the subject being treated. The therapeutic effect may be objective (i.e. measurable by tests or markers) or subjective (i.e. the subject gives an indication of an effect or an effect). An effective amount may vary from about 0.01 μg Kg to 5000 μg Kg, alternatively from about 0.1 to 1000 μg Kg, alternatively from about 1 to 500 μg Kg. The actual amounts will also vary depending on the route. administration, the use or otherwise of in vivo transfection method, the size and weight of the subject, as well as the possibility of co-use with other agents.

In the context of the invention, the mode of administration of the nucleic acid according to the invention, or of the vector according to the invention, or of the immunogenic or vaccine composition according to the invention can be carried out intravenously, under cutaneous, intradermal, intramedullary, or intramuscular.

The invention will be explained in more detail by the following examples, without limiting its scope.

DESCRIPTION OF FIGURES

Figure 1: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 2.

Figure 2: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 4.

Figure 3: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 5.

Figure 4: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 6.

Figure 5: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 7.

Figure 6: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 1.

Figure 7: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 12. Figure 8: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 13.

Figure 9: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 14.

Figure 10: Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 15.

Figure 11: Schematic representation of the CAEV genome construction, pSHIV _KU2 plasmid, pCA-LTR-SHIV _KU2 IN- and pA4SHIV _KU2 -

Figure 12: Evaluation of the number of T cells secreting IFN-γ Balb / c mice. Spleen cells of BALB / c immunocompetent mice that were immunized with pA4SHIV _KU 2 and pCA-LTR-SHIV _KU 21 N- and stimulated with the Gag, Env peptides and the Tat + Rev + Nef peptide pool. The number of spots has been calculated for 1 million PBMCs. Figure 13: Evaluation of the number of human T cells secreting IFN-γ in immunized NOD / SCID-hu mice. Spleen cells from immunodeficient mice reconstituted by human blood mononuclear cells and immunized with pA4SHIV _KU 2 or pCA-LTR-SHIV _KU 2I N- or pSHIV _KU 2 are stimulated by the Gag, Env peptides and by the Tat + Rev peptide pool + Nave. The number of spots was calculated for 1 million PBMCs and normalized to 20%.

Figure 14: Representation of CA-LTR-SHIV _KU 2-IN- vector preparation.

Figure 15: Schematic representation of the vector CA-LTR-SHIV _KU 2-

Figure 16: Schematic representation of the vector CA-LTR-SHIV _KU 2-IN-.

Figure 17: Representation of the preparation of the vector CAL-HIV-IN-.

Figure 18: Schematic representation of the CAL-HIV-IN- vector. SEQUENCE DESCRIPTION

SEQ ID NO: 1 represents the sequence of a chimeric retroviral genome comprising the LTRs of the SAEV and the genome of the SHIV deleted sequences encoding the integrase.

SEQ ID NO: 2 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-1.

SEQ ID NO: 3 represents the CAEV LTR sequence.

SEQ ID NO: 4 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of SIV.

SEQ ID NO: 5 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-2.

SEQ ID NO: 6 represents the sequence of a chimeric retroviral genome comprising the CAEV LTRs and the FIV genome. SEQ ID NO: 7 represents the sequence of a chimeric retroviral genome comprising the CAEV LTRs and the SHIV genome.

SEQ ID NO: 8 represents the sequence of the SIV integrase.

SEQ ID NO: 9 represents the sequence of the integrase of HIV-1.

SEQ ID NO: 10 represents the sequence of the integrase of HIV-2.

SEQ ID NO: 11 represents the sequence of the FIV integrase.

SEQ ID NO: 12 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-1 deleted from the sequences encoding integrase.

SEQ ID NO: 13 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-2 deleted from the sequences encoding integrase.

SEQ ID NO: 14 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of FIV deleted sequences coding for integrase.

SEQ ID NO: 15 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the SIV genome deleted of the sequences encoding integrase.

SEQ ID NO: 16 represents the sequence of the vector pCA-LTR-SHIV _K u2-

SEQ ID NO: 17 represents the sequence of the vector pCA-LTR-SHIV _KU 2-IN-.

SEQ ID NO: 18 represents the sequence of the vector CAL-HIV-IN-.

EXAMPLES

1. Material and methods

1.1. Vaccine vectors (Figure 1)

1 .1 .1. DCA-LTR-SH IVKMP and DCA-LTR-SH Vectors IVKMP-I N-

The CA-LTR-SHIV _K U2 vector contains the Simian and Human Immunodeficiency Virus (SHIV) genome deleted from the SIV LTRs and replaced by the CAEV LTRs. The SHIV contains a chimeric genome composed of that of the VIS-mac239 in which the tat, env and SIV genes were deleted and replaced by the HIV-1 vpu, tat, env and rev genes. The vector therefore carries the SIV vp, vpx, gag, pol, SIV genes and the tat, rev, vpu and env genes of HIV-1 under the transcriptional control of the 5 'and 3' LTRs of CAEV. The pol gene has been deleted from the coding sequences of the integrase (in). The non-deleted pCA-LTR-SHIV _KU 2 vaccinating vector of the integrase coding sequences consists of the sequence SEQ ID NO: 16 (FIG. 15). The pCA-LTR-SHIV vector _KU 2 -IN-, deleted coding sequences of the integrase consists of the sequence SEQ ID NO: 17 (Figure 16).

The construction of the CA-LTR-SHIV vector _KU 2-IN- was carried out as follows (FIG. 14). The SHIV- _K U2 vector was digested with EcoR1 and Nar1, and then the 0.8 kb LTR fragment was removed. The vector CAEV-pBSCA was then digested with EcoR1 and Nar1 and the 0.5 kb LTR fragment was purified. Both fragments were then ligated. The SHIV-1 LTRCA vector was then digested with Stu1 and Aval and the 0.8 kb LTR fragment removed. The 3 'LTR of CAEV was amplified with Stu1 and Aval primers, the PCR products were digested with Stu1 and Aval and the 0.5 kb LTR fragment was purified. Both fragments were ligated to generate CAL-SHIV _K u2- Finally digestion with Kpn1 and Acc1 in the pol gene was performed to remove 314 bp of the SHIV integrase gene to generate CAL-SHIV

1 .1 .2. PSI-I IV ^ and A4SH IVKMP vectors

Plasmids pSHIV _KU 2 and pA4SHIV _KU 2 are plasmids used as controls. Their constructions have been described in many publications (Liu ZQ et al., 2006, Ramakrisna Hegde et al., 2005). 1.2 Production of vaccine DNA

1 .2.1. Bacterial culture

Bacteria E. co // ^'K12 (JM109) containing the plasmid are being pre-cultured in 5 ml of LB medium containing 0.05 mg / ml kanamycin and incubated overnight at 30 ° C with stirring at 150 rev / min (rpm). From the pre-culture, the bacterial suspension is diluted 100 000th in LB medium, then 50 μl of the dilution are spread on the surface of the agar / LB / Kanamycin contained in a petri dish which is then incubated at 32 ° C overnight. The isolated colonies grown on the agar of the petri dish are inoculated in 5 ml of LB liquid medium containing 0.05 mg / ml of kanamycin and cultured with stirring at 150 rpm at 30 ° C overnight. A fraction (1 ml) of the culture is used for rapid extraction of the DNA using the Macherey-Nagel Mini-prep kit or Qiagen, according to the recommended protocol, and the extracted DNA is separated on a gel. agarose at 1% to check its quality. The bacteria corresponding to the DNA deemed satisfactory are used to seed the 1 L cultures which are cultured under the same conditions as above, for the isolation of DNA in maxipréparation.

1 .2.2. Maxi-preparation: plasmid extraction

Bacteria cultured with stirring (150 rpm) overnight are pelleted by centrifugation (4000 g, 4 ^< C, 15 min) and the pellet is resuspended in 8 ml of resuspension buffer (Tris-HCl 50mM pH8). , EDTA 10mM). The cells are then lysed by addition of 8 ml of alkaline lysis buffer (200 mM NaOH, 1% SDS). to release the plasmid DNA. The lysate is neutralized by addition of 8 ml of neutralization buffer (3M potassium acetate pH 5.5). The mixture is then incubated for 5 min on ice and then centrifuged 15 minutes at 15 000 g at 4 ^q C. The solution containing the DNA is transferred to a pre-equilibrated column to retain the plasmid DNA. The column is washed three times with the wash buffer and the DNA is then eluted and precipitated with isopropanol. The precipitated pellet of DNA is obtained by centrifugation (30 min, 15,000 g at 4 ° C). The DNA is then washed with 2 ml of 70% ethanol and centrifuged for 10 min at 4 ^< Ό to 15 000 g to remove excess impurities and salts and the pellet is dried and resuspended in a suitable volume of ultrapure water. .

The concentration of the DNA solution is then determined spectrophotometrically at a wavelength λ equal to 260 nm and the quality of the plasmid is verified by electrophoretic migration on a 1% agarose gel. The size of the plasmid and the integrity of the plasmid are verified on agarose gel after digestion with Bam H1 and Eco R1 restriction enzymes, for example.

1 .2.3. Verification of the pCA-LTR-SHIV _K np-IN- plasmid by enzymatic digestion

An aliquot of 0.5 micrograms of the plasmid is subjected to digestion with 2 units of EcoR1 enzyme BamH1 and Sph1 for 60 minutes at 37 ^<Ό in buffer 1 X appropriate for each enzyme, and in a final volume of 20μΙ. The profile of the digestion is verified by electrophoretic migration in a 1% agarose gel with a 1 × TAE buffer and revealed by ethidium bromide (BET) and observation of the gel under UV.

1.3 Cell cultures and treatments:

1 .3.1. Cellular models and crop conditions

The cell lines were obtained from the National Institutes of Health

AIDS Research and Reference Reagent Program in the United States. The cells are cryopreserved in 10% dimethylsulfoxide (DMSO), at -170% in liquid nitrogen. They are thawed and then cultured in culture flasks.

HEK293T cells (Immortalized Human Embryonic Kidney 293) are a permanent line of human embryonic kidney cells. They are used because they are very easy to transfect, with very high transfection efficiencies that can reach 100%. The lentiviral genome is strongly expressed in these cells and the proteins assemble into infectious particles and their co-culture with the indicator cells (CEM or M8166) allows the formation of typical syncytia. The HEK293T cells are adherent, grown in monolayer on the surface of the flasks in MEM medium supplemented with 10% fetal calf serum (FCS), 1% penicillin 5,000 Unit / ml streptomycin 5,000 μg / ml and 1% gentamycin 10 mg / ml. The cells are maintained at 37 ^<C in a humid atmosphere of 5% C0 _2. The culture medium is changed every three days. To carry out subcultures, the nutrient medium is removed, the cells are washed with PBS / EDTA and incubated for 1 minute at 37 ° C in the presence of 0.5% trypsin-EDTA 0.01%. After detachment of the cells, a volume of MEM medium is immediately added to the cells and then the cells are homogenized and transferred to new flasks.

CEMx174 and M8166 cells are human CD4 + T cell lines that are permissive to human and simian lentivirus infection and form typical cytopathic effects (CPE). They are non-adherent and are cultured in RPMI medium supplemented with 10% FCS, 1% penicillin-streptomycin and 1% gentamycin. The cells are maintained at 37 ^<C in a humid atmosphere of 5% C0 _2. The medium is changed every three days by performing a centrifugation step at 1500 g for 5 minutes. The pellet is then resuspended by suctions and successive upsets in a suitable volume of culture medium.

1 .3.2. Functional evaluation with in vitro bioassay

Transfection of HEK293T with pCA-LTR-SHIV _KU2 - \ ¼- or pSHIV _{KU2 plasmids}

The transfection method used is the method with ExGen500. ExGen500 (Euromedex, France) is composed of a cationic polymer based on linear polyethylenimine. This polymer has a very high cationic charge density for forming complexes with DNA by ionic bonds. These ExGen500 / DNA complexes are then able to interact with the plasma membranes of generally anionic cells (interaction via sulphated proteoglycans). This results in endocytosis of the complexes by the cells and their transport to endosomes / lysosomes. By its ability to protonate at acidic pH, ExGen500 buffers the acidic vesicle medium thus preventing degradation of the transfected DNA. This property also causes osmotic shock, which allows the DNA to be released into the cytoplasm of the cell. ExGen500 then promotes the transport of DNA to the nucleus and prevents its degradation by cytoplasmic nucleases.

Five μg of the plasmid DNA are added to 350 μl of 150 mM NaCl solution and 15 μl of ExGen 500. The mixture is incubated for 40 minutes at room temperature. The mixture is then added to the flasks containing HEK-293T coated with freshly renewed medium.

Infection of CEMx174 and amplification of viral stock The transfected HEK-293T DNA pCA-LTR-SHIV _KU2 -in- or pSHIV _KU2 are being co-culture with CEMx174, and from 48 hours the CEMx174 develop signs of infection resulting the formation of ECP that result from the fusion of CEMx174 to form syncitia. Infected CEMx174 are transferred to a new flask in the presence of fresh CEMx174 to amplify the virus that is harvested from the supernatant.

Viral production

The viral stock is collected from 48 hours using a syringe, then the cell debris is removed by passing through a 0.22μηι diameter filter before being put in tubes that are stored. at -80 'C.

Inoculation of cells with the virus

An aliquot (10-100 μΙ) of the virus supernatant is used to inoculate the target cells to evaluate its infectivity by cytopathic development or by detection of expression of marker genes.

Titration of the virus on non-adherent cells

The supernatant containing virus was diluted every ten (serial dilutions) in medium to obtain dilutions of 10 ^{~ 1-10} ^{~ 6} which are used to inoculate quadriplate in wells containing 1 .10 ⁵ cells per well in 0, 5 to 1 ml of RPMI medium in a 24-well plate. The cells thus inoculated are incubated at 37 ° C. and 5% CO ₂ and maintained by changing the medium every 3 days. They are observed regularly for the development of ECP.

1.4. Electron microscopy of HEK293T cells transfected with pCA-LTR-SHIVKU ₂ -I N-

In order to examine whether the proteins produced by the vaccinia genome pCA-LTR-SHIV _KU 2-IN- assemble into viral particles, the inventors carried out morphological studies by electron microscopy (EM).

Samples of HEK293T cells transfected with pCA-LTR-SHIV _KU 2-IN-, pSHIV _KU 2 or A4SHIV _KU 2 are fixed in a solution of 2.5% glutaraldehyde diluted in a cacodylate buffer (0.1 M sodium cacodylate). . They are then post-fixed at 4 ° C in cacodylate buffer containing 1% Osmium tetraoxide (OsO4) for 60 minutes. The samples are then incubated overnight at 4 ^<C in the dark in uranyl acetate pH 4. The samples were then immersed in baths successive 10 minutes of ethanol diluted to 30%, 60%, 90% and 100% respectively. Then the samples are immersed for two hours in a 50/50 mixture of pure ethanol and epoxy resin (8 ml of DDSA, 7 ml of MNA, 13 ml of Epoxy). The samples are then placed for two hours in pure epoxy resin before being included in Beem capsules and polymerized for 48 hours at 60 ° C.

Ultrafine sections of these areas are made using a diamond knife using an ultra microtome. These 70 nm thick sections are deposited on copper grids to be observed at a voltage of 80kV using a Jeol 1200 EX transmission ME.

1.5. Immunization of the mice with the pCA-LTR-SHIV _K u2-IN- vaccinia DNA.

1 .5.1. Humanization and vaccination of NOD / SCID mice

The 6 week old mice are irradiated with a dose of 120 Centigray of gamma radiation for 50 seconds.

Humanization of mice with PBMCs of human blood

Whole blood collected on sodium citrate is centrifuged (2000g, 10 min, 20 ° C) to recover the white cell layer between plasma and red cells. The cells are diluted 3 times in PBS / EDTA, gently deposited over a Ficoll cushion (lymphocyte separation medium) and then centrifuged for 45 minutes at 2000g at 20 ° C. The PBMC are recovered, washed several times in PBS / EDTA and resuspended in PBSxl then 50.10 ⁶ PBMC in 0.1 ml are injected for each mouse intraperitoneally. Immunization of mice

After 48-72h post-humanization, the SCID-hu mice are injected intramuscularly (IM) with 50 .mu.g of DNA pCA-LTR-SH IV _KU2 -in-, pSHIV _KU2 or pA4SHIV 2- _KU BALB / Those aged 6-8 weeks are directly immunized by IM injection with 100 μg of each of the DNAs.

1 .5.2. Method of evaluation of the humoral response

Abnova Sandwich ELISA Test:

This test is based on the detection of antibodies against viral antigens that are attached to the bottom of wells of the 96-well plate. The sera to be tested (recovered at different post-immunization times), the positive and negative controls are deposited in the wells. The anti-HIV antibodies possibly present are fixed on the viral antigens. After several washings of the wells to eliminate the excess and non-specific bindings, a secondary detection acid is added which carries a biotin molecule which interacts with streptavidin coupled to the HPRO enzyme. The antigen / Ac complexes formed will then be detected by adding the substrate of the enzyme, TMB, which will give rise to a color reaction. The coloration is translated into optical density by reading with an ELISA reader photometer.

The reagents and products are brought to room temperature. The negative and positive controls (1 ΟΟμΙ) provided in the kit, the blanks (1 ΟΟμΙ) and the serum samples 10 and 50 μΙ are deposited in the wells in a final volume of 10ΟμΙ. The plate is incubated for 30 minutes at 37 ° C, then rinsed with the wash solution. The solution of secondary Ac diluted to one hundredth is added (100 μΙ) in all the wells except in the whites. The plate is then covered with parafilm and incubated for 20 minutes at 37 ^< Ό. A solution A and B of TMB is added after a washing step and the plate is incubated for 15 min at laboratory temperature. To stop the staining step, 100 μl of H ₂ SO ₄ to ₂ N are added per well. The reading of the plate is made at 450 nm.

Samples having an absorbance value equal to or greater than the threshold value are considered as positive, the threshold value being determined according to the following formula: Cutoff Value = NCx + 0.100 with NCx = the average of the absorbance values of the two controls negative.

Sero-neutralization test

This technique is based on the ability of seroneutralizing antibodies (sero-N) to inhibit the infection of virus-sensitive cells. For the detection and evaluation of serum Ac sero-N, a constant amount of infectious virus is contacted with serial dilutions of the serum to be tested, then the mixture is inoculated into a microplate-permissive cell culture and incubated at 5 days. The virus is most often a cytopathogenic strain, and therefore the absence or reduction in the number of PCEs translates the presence of Ac into the tested serum.

The SHIV _K U2 viral stock is diluted 1/1 in RPMI (the volume of virus supernatant is determined per 100 TCID ₅₀ , which corresponds to the dilution of virus for which 50% of the wells have syncitia) and the sera recovered from the mice. NOD / SCID controls, humanized and vaccinated with pCA-LTR-SHIV _KU 2-IN- or pSHIV _KU 2 are diluted in the same medium (at dilutions 10, 20, 40, 80, 160 and 320). The diluted virus and serum dilutions were mixed in a 96 well plate (1 ΟΟμΙ / well) and the mixture was incubated 1 h at 4 ^<Ό. The mixture is then deposited on cells M8166 (1 .10 ⁵ cells / well) previously cultured in a 24-well plate. In this experiment, the positive control is represented by the virus from serum-free pSHIV _K u2 and the negative control corresponds to cells alone. Evaluation method of cellular response: Elispot test

This test aims to highlight and evaluate the proportion of T lymphocytes (LT) that secrete IFN-γ in specific response to antigenic stimulation.

The mice are deeply anesthetized before the whole blood and the spleen are removed. The spleens of the mice are put in RPMI medium in ice. The blood in dry tubes is used to isolate the serum. The splenocytes are isolated following grinding of the spleen in a petri dish between a pair of slides in the presence of PBS and 1% EDTA. The cells are washed twice in PBS / EDTA (2000g centrifugation, 5 min at 20 ° C.) in order to purify and enrich in splenocytes. Before inoculating the wells with the cells, the plate is washed with PBS and incubated for 30 min with PBS + 10% FCS at laboratory temperature. The cells ( ⁵ × 10 ⁵ splenocytes) are inoculated in each well, and are inoculated with peptide pools of the Gag, Env, Tat, Rev and Nef proteins at a final concentration of 2 μg / ml. Positive controls (CD3-2 included in the kit) and negative controls are added to the test. The plate is covered with an aluminum pouch and incubated at 37 ° C for 19 hours. The cells and peptides are washed, then the biotinylated anti-IFN-γ monoclonal Ab (7-b6-biotin) is added, and the plate is covered and incubated for 2 hours at room temperature. Streptavidin diluted in PBS containing 0.5% FCS is added (1 μM / well) and the plate is incubated for 1 hour at room temperature. The TMB, developer substrate, is added later after another washing step, then the plate is washed and dried after emergence of blue spots. The reading of the plate is done with the binocular magnifier at magnification ^* 40.

The criteria for positivity of a well are determined for each condition by calculating the average of the number of spots of the duplicates, as well as the standard deviations. The number of spots is calculated for 1 million PBMC and is normalized to 20% for the data obtained in NOD / SCID mice. The test is considered to be positive if the value of the average of the spots is greater than 10 spots per million PBMCs which corresponds to the average of spots obtained with the culture controls.

1.6. Phenotypic and functional examinations of antigen-specific T cells by flow cytometry

1 .6.1. Isolation of peripheral mononuclear cells Mononuclear cells of human peripheral blood are prepared as indicated above. The splenocytes of mouse spleens isolated according to the protocol described above are also resuspended in serum-free AIM V medium for culture and cytometry tests.

1 .6.2. Antigenic stimulation and cell culture

To examine whether antigen-specific cells are able to proliferate and produce cytokines and lytic molecules, splenocytes and PBMCs are labeled with CFSE (1 μg ml) for 10 min at 37 ^< C, then the cells are washed with 1 X PBS to remove excess. The labeled cells are seeded in deep wells of 96 well plates at 2.10 ^s / well in 1 ml of AIM V medium, then stimulated with the different peptide pools (Gag, Env and Tat + Rev + Nef) at a rate of of 2 μg / ml in the presence of anti-CD49 and CD28 co-stimulation mAbs. Cells without peptides are used as a negative control and cells supplemented with phytohemagglutinin (PHA) 2 μg / ml are used as a positive control. The cells are cultured for 5 days (37 ° C. under humidity) and then restimulated with the same peptide pools for 6 hours before labeling them. The cells are harvested by centrifugation (2000g, 5 min, 4 ° C), resuspended in 100 μl of PBS and first labeled with surface Ab (CD3, CD4 and CD8) [Pacific Blue anti-human CD3 (5 μl) ), Anti-human CD4 PE (10 μl) and APC / Cy7 anti-human CD8 (10 μl) for 30 min at room temperature. The cells are then centrifuged and washed with 1 X PBS and then fixed and permeabilized in 100 μl of Cytofix Cytoperm BD. The cells are then incubated for 20 minutes at 4 ^<C with Ac "anti-human IFN-γ PE-Cy7" and "Alexa Fluor 647 anti-human Granzyme A" (5 μΙ) for cytoplasmic markings. The cells are finally washed with PBS and fixed with 4% PFA prior to acquisition and flow cytometric analysis.

1 .6.3. Instrumentation

An LSRII flow cytometer from BD connected to BD FACSDiva6 software was used. This instrument can measure up to 13 fluorescence parameters and two physical parameters such as FSC size (Forward Scatter) and SSC (Side Scatter) complexity or granulosity. The instrument is equipped with three lasers. The blue laser emitting at 488nm can independently excite several fluorochromes (FITC, PE, PE-Cy7). The red laser emitting at 633nm can excite the APC and APC-Cy7 fluorochromes. And finally the purple laser that emits at 405nm can excite the Pacific Blue fluorochrome. 1.7. Immunization of macaque

A total of 12 cynomolgus macaques are used in this study. Six macaques constitute the control group and the six others the vaccinated group. The animals were immunized by a single double injection of DNA by the intramuscular route (4 mg / animal) and 1 mg / animal by electroporation (EP).

The reasons for this strategy of immunization with a single dose of the vaccine are multiple. One of the main reasons is not to disturb the generation, maturation and amplification of memory T cells by the primary effector T cells associated with each re-immunization step.

Immunized animals were followed longitudinally (once a week during

4 weeks then 1 week out of 2 until week 32, then 1 week out of 4 for 10 months to examine the immune responses induced by the vaccine. Blood samples taken at weeks -2 and -1 prior to immunization were used to examine possible baseline responses. Peripheral blood mononuclear cells (PBMC) are isolated and used to evaluate T-cell responses by ELISPOT IFN-λ, surface and intracytoplasmic markings, and flow cytometric analysis.

2. Results

2.1. Qualitative and quantitative verification of the construction of plasmid pCA-LTR-SHIV _KU 2-IN-

2.1 .1. Presence of plasmid PCA-LTR-SH IVKMP-IN-

When the plasmid DNA was isolated from a polyclonal bacterial culture obtained from a preculture in LB medium used to seed a large volume of liquid LB medium, a significant proportion of episome is observed around 2000bp. The electrophoretic profile also demonstrates the presence of a single DNA band around 14,000 bp (which in theory is 13,739 bp) corresponding to the plasmid.

When the DNA is isolated from a bacterial culture carried out first on a petri dish to obtain isolated colonies, these colonies having been used for preculture and the mass culture serving for the isolation and purification of the Plasmid DNA, the electrophoretic profile obtained after separation of 0.5 μg of DNA shows the absence of an episome and shows three bands of high molecular weight DNA, corresponding to the circular, wound and supercoiled forms of the plasmid pCA-LTR- SHIV _KU 2-IN-. The purity and quantitative evaluation of the plasmid DNAs of our two preparations were verified by spectrophotometry. The values of the measurements of the absorbance at wavelengths 230, 260 and 280 were used to determine the ratios 260/280 which were 1.75 and 1.82 and 260/230 of 2.04 and 1.92 respectively. a satisfactory quality of our DNA. The DNA concentrations are respectively 545 μg ml and 765 μg / ml.

2.1 .2. Enzymatic digestion of DCA-LTR-SH IVKMP- IN-

The profile of the digestion of the plasmid with EcoR1 reveals the presence of two bands of approximately 5000 and 7500 bp resulting from the cuts at the two EcoRI sites, three bands with Bam H 1 of 2400 bp, 4900 bp and 7400 bp resulting from the cuts at the two Bam H sites 1, and a band with Sph 1 located at 10 000pb resulting from the cut at the single site Sph1. An additional 2400 bp band is also observed with BamH 1 digestion and it would result from the episome.

2.2. Evaluation of the functionality: Effect of plasmid sur on the cells

The HEK293T cells were transfected with a control plasmid pCG-GFP expressing GFP, the plasmid pSH IV _KU 2, and then with the plasmid pCA-LTR-SH IV _KU 2-IN-. Cells transfected with the plasmid pCG-GFP made it possible to estimate the transfection efficiency by evaluating the number of GFP + cells.

To verify that EK293T H cells transfected with pCA-LTR-SH IV _K u2-IN- or pSH IV _K u2 produce virions that induce typical syncitia, these cells were co-cultured with CEMx174, followed by appearance of CPE was followed by observation under the microscope. Both co-culture produced characteristic ECPs.

To verify that the SH IV _K U2 virus produced by the transfected cells replicates several times, the supernatant of the transfected cells was used to infect the human CD4 + T lymphocyte line M81 66. The results obtained with SH IV _KU 2, clearly show that It infects and induces PCEs especially with the highly permissive cell line M81 66. These PCEs appear as early as 48 hours but also at late stages.

Then, to verify that the pCA-LTR-SH IV _K u2-IN- vaccine vector DNA allows only a single replication cycle (as it is deleted from the in gene), the recovered supernatant containing the CA-LTR- SH IV _KU 2-IN- was inoculated once, then a second time to M81 66 in culture. The first inoculation produced CPE from 48 hours and after 76 hours they were more numerous. The supernatant of these infected cells was recovered and inoculated again with M81 66. In contrast to the previous inoculation, no CPE is visible either at 48 or 76 hours postinoculation. These results make it possible to conclude that pCA-LTR-SHIV _K u2-IN- allows the target cells to be transduced once in culture.

The presence of typical ECP, suggests that plasmid DNA is replicated in HEK293T cells and produced virions CA-LTR-SHIV _KU 2-IN-. The PCEs of the first infection are indicative of the infectivity of the M8166 human CD4 + LT line by the particles produced, and their absence during the second infection is indicative of the productive replication deficiency in these cells.

2.3. Electron microscopy analysis of the morphogenesis of viral particles in HEK293T cells transfected with PCA-LTR-SHIVKUS-IN-

It was then determined whether the proteins produced by the vaccinia genome pCA-LTR-SHIV _KU 2 -IN- correctly assembled into viral particles in HEK293T cells. The morphology of HEK293T cells transfected with plasmid pCA-LTR-SHIV _KU 2-IN- was examined by ME.

The results showed that the viral particles are present on the surface of the cells in the form of buds and mature viral particles which have detached themselves from the cells. Thus, the viral proteins of the pCA-LTR-SHIV _K u2-IN- vaccine assemble to give viral particles that bud out of the cells. 2.4. In Vivo Vaccine Test on Humanized SCI P Mice and BALB / c Mice

2.4.1. Evaluation of humoral response in SCID-hu mice vaccinated with DCA-LTR-SH IVKMP-I N-

Serum samples from immunized mice taken at approximately 1 month post-immunization are examined for the presence of Ac that binds specifically to the virus antigens using a commercial ELISA. The results of this analysis are summarized in Table 1 (below). These results show that about half of the samples from mice immunized with the pCA-LTR-SHIV _KU 2-IN- DNA, like those immunized with SHIV _KU 2 DNA, have lower levels of positive ( 44% and 37% respectively) in contrast to those from mice immunized with pA4SHIV _KU 2 (50%). These results demonstrate the ability of pCA-LTR-SHIV _K u2-IN- vaccine DNA to induce humoral responses in NOD / SCID-hu mice. One in three serum samples from mice immunized with pA4SHIV _KU 2 had a higher OD value than those obtained with sera from mice immunized with pCA-LTR-SHIV _KU 2-IN- and pSHIV _KU 2- This plasmid is non-replicative, the proteins of the virus remain associated with the membrane of the transfected cells and therefore should induce less Ac.

SCID-hu and SCID-hu immunized with pA4SHIV _KU2 , pCA-LTR-SHIV _KU2- IN- and pSHIV _KU2 and controls.

2.4.2. Neutralizing activity analysis by serum neutralization

Sera of SCID-hu mice vaccinated with the selected pCA-LTR-SHIV _K u2-IN- or SHIV _KU 2 plasmids are those whose OD has been found positive by ELISA. Due to the small amount of serum, some samples with high OD values for the ELISA could not be examined by serum neutralization. A serum sample of SCID-hu mice and vaccinated with pCA-LTR-SHIV _KU 2-IN- found negative was also used for the ELISA assay to ensure the reliability of the test.

Table 2: Analysis of the neutralizing activity of the sera of the immunized mice. Dilutions (1/10, 1/20, 1/3, 20) were mixed with SHIV _KU2 virus (100 TCID ₅₀ ), incubated and then used to inoculate M8166 cells. After 5 days postinoculation, the induced EPCs are enumerated and used to evaluate serum neutralizing activity. Legend: +++ correspond to a strong neutralization, ++ quite high neutralization, + less neutralization, - no neutralization action. For CA-LTR-SHIV _K u2, two types of samples are shown, one type of sample having a less neutralizing profile compared to the other two samples.

The number of CPEs obtained was higher in M8166 cells infected with serum free SHIV _K U2 virus (76 ECP) or with non-immune mouse serum (42 ECP) (negative controls), which allowed us to to set the viral neutralization negativity threshold at 42 ECP (data not shown in the table). On the other hand, the number of ECP becomes almost zero when the virus is incubated with serum diluted 1/10 of the mice immunized with CA-LTR-SHIV DNA _K U2-IN- or SHIV _K U2- This number increases as serum dilution is increased indicating a dose effect. For example, with the serum of a mouse immunized with pCA-LTR-SHIV _K u2-IN- at the 1/10 dilution, 5 ECP is obtained, whereas at the 1: 320 dilution a value of 69 ECP is obtained. similar to serum free control.

2.4.3. Evaluation of the immunogenicity of PCA-LTR-SH IVKMP-I N- in vaccinated BALB / c mice

To study the immunogenicity of the pCA-LTR-SHIV _K u2-IN- vaccine in BALB / c mice, the animals were injected with a single dose of 100 μg of DNA intramuscularly. The proportion of spleen cells (splenocytes) specific for the antigens was examined by ELISPOT. The results of these viral and IFN-γ secreting studies demonstrate the ability of the DNAs used to induce specific immune responses directed against all the antigens studied (Figure 12). The cellular T responses with the plasmid pA4SHIV _KU 2 are twice as high as with the plasmid pCA-LTR-SHIV _KU 2-IN-. This small difference in efficacy between the two DNAs could be related to a better quality of pA4SHIV _KU2 DNA than pCA-LTRSHIV _KU 2-IN-. These results nonetheless make it possible to know the basic level of the immune responses induced by these vaccines in normal mice and make it possible to compare them with the immune responses obtained in SCID-hu mice.

2.4.4. Evaluation of the cellular response in NOD / SCID-hu mice immunized with the different DNAs. The NOD / SCID-hu mice were immunized by intramuscular injection with a single dose of 50 μl of CA-LTR-SHIV DNA _KU 2-IN-, A4SHIV _KU2 or SHIV _KU 2, then the splenocytes were used to examine the immune response by ELISPOT to evaluate the proportion of antigen-specific cells producing IFN-γ. As shown in FIG. 13, there is a large number of T cells producing human INF-γ in response to stimulation by Gag, Env or Tat + Rev + Nef antigens in the form of SIV or HIV peptides. . It is interesting to note that the responses obtained after immunization with pCA-LTR-SHIV _KU 2-IN- are almost similar to those obtained with pSHIV _KU 2 and both of which are very much higher than those obtained after immunization with pA4SHIV _KU 2- The predominance of responses induced by pCA-LTR-SHIV _KU 2 is against Gag and Tat + Rev + Nef antigens.

All these results demonstrate that the pCA-LTR-SHIV _KU 2 DNA is highly immunogenic in NOD / SCID-hu mice and that it preferentially induces responses against the antigens known to be associated with the protection against parasites. pathogenic viruses.

2.5. Phenotypic and functional examinations of antigen-specific T cells by flow cytometry

2.5.1. Monomarguaqe performed at day zero (made the day of recovery of mouse spleens)

These mono-markings serve on the one hand, to examine that the selected antibodies detect the targets well, and on the other hand to evaluate the presence and proportion of human cells in the spleens of SCID-hu mice.

Unlabelled lymphocytes from humanized and non-humanized mice were analyzed by flow cytometry to measure basal cell fluorescence (negative control).

Detection of CD3 + LT is performed with human anti-CD3 antibody coupled to fluorochrome "Pacific Blue". These cells peak at 10 ² in the profile of isolated cells in SCID-hu mice vaccinated with plasmid pCA-LTR-SHIV _KU 2-IN-. This peak is not present in the cells recovered in non-hued SCID mice.

For mono-labeled cells with anti-CD4 + Ab (PE anti-human CD4), either for the control or for our sample tested, no significant peak was observed. This would be due to the anti-CD4 Ab used that would be non-functional.

For the detection of CD8 + LTs, a human monoclonal anti-CD8 antibody coupled to APC / Cy7 fluorochrome was used. It allows the highlighting of a peak located between 10 ² and ³ in the profile of isolated cells in SCID-hu mice vaccinated with pCA-LTR-SHIV _K U ₂ -IN- and not in non-hued SCID mice.

The proportion of lymphocytes producing the GRA molecules could not be evaluated because no peak difference was observed on the cells from immunized and non-immunized mice labeled with anti-GRA A coupled to Alexa Fluor 647.

On the other hand, the cells labeled with the human anti-IFN-γ coupled to the fluorochrome PE-Cy7 showed a sharp peak situated at 10 ² with the SCID-hu mouse cells vaccinated with pCA-LTR-SHIV _KU2- IN- which is absent with the cells of non-immunized non-hu SCID mice.

2.5.2. Phenotypic and T cell functions in immunized animals.

To examine the phenotype and functions of T cells specific for virus antigens, CFSE-labeled cells are incubated for 5 days with the peptides (Gag, Env and Tat + Rev + Nef) and then re-stimulated for 6 hours with the cells. same peptides. The cells are then labeled with Ac and examined as above.

The results of this analysis show the presence of CD3 + and CD8 + cells which produce IFN-γ and which have GRA molecules especially with cells from vaccinated NOD / SCID-hu mice. Indeed, at least 6% of the CD8 + LTs produce IFN-γ and 1.7% produce the GRA A in immunized NOD / SCID-hu mice, compared to only 2.5% and 0.6% respectively in the control mice. These results demonstrate the presence of activated CD3 + CD8 + cells, producing GRA and IFN-γ, which corresponds to an effector cellular immune response induced by our pCA- vaccine.

2.6. Analysis of immune and humoral responses in macaques

2.6.1. Analysis of immune responses of T cells by ELISPOT IFN-A test

A fraction of the mononuclear cells isolated from the collected blood samples was used to evaluate the proportions of IFN-λ secreting cells in response to stimulation by the Gag, Pol, Env and Tat + Rev + Nef viral antigens at the same time. using a commercial kit. The results of the first 20 weeks of analysis are shown in Table 3. They clearly demonstrate that following a single administration of the vaccine vector CAL-SHIV-IN-, all animals developed cellular immune responses characterized by antigen-specific cells, which secrete IFN-λ. These responses are heterogeneous depending on the animals that have MHC-1 haplotypes different from each other. The immune responses are characterized by the presence of a first peak of primary response at 2-4 weeks post-immunization (PI), then later responses from 8-10 weeks PI, and this in the absence of a second immunization. It is very interesting that the intensity of the second peak is often much larger than ¹ peak especially for the animal BX80 where the number of cells secreting IFN-λ is multiplied by 3.

Post-Immunization Week

1 2 3 4 6 8 10 12 14 16 18 20

Animal Ag Number of spots / million PBMC in response to antigens (Ag)

Gag 208 821 666 472 317 504 538 474 226 574 621 862

Pol 0 105 17 350 15 32 0 97 27 1 1 1 1 168

BX80 Approx. 0 84 1 7 124 625 753 67 36 41 48 132

TRN 0 64 0 107 137 1238 1663 1084 728 1215 800 1752

Gag 0 565 1033 443 147 0 488 377 69 152 96 298

Pol 0 33 75 193 0 0 340 145 19 0 0 12

Send 0 87 49 72 0 0 1035 60 40 25 9 20

BX83

TRN 0 51 204 1 16 0 0 731 393 195 224 143 432

Gag 0 1279 703 752 300 380 161 427 155 461 91 1269

Pol 1 88 36 24 15 5 8 80 0 19 9 44

BX84 Approx. 1 340 85 105 48 57 21 72 13 96 57 217

TRN 0 205 73 128 27 416 417 761 59 272 79 228

Gag 16 388 1989 1220 813 885 840 1795 0 85 472 1344

Pol 0 205 17 45 8 0 13 0 0 0 7 120

BX72 Approx. 7 79 57 56 12 168 56 5 0 0 12 132

TRN 5 35 161 192 92 692 445 736 0 25 1 19 587

Gag 8 668 163 213 45 52 76 555 24 83 80 227

Pol 0 548 60 20 0 31 0 104 132 1 0 59

BX78 No. 0 491 68 97 437 181 0 259 24 15 19 73

TRN 3 296 279 39 0 84 824 685 17 69 39 275

Table 3: Summary of interferon gamma cytokine secretory T cell (IFN-λ) ratios in response to stimulation by viral antigens (Gag, Pol, Env and Tat + Rev + Nef) expressed by the vaccine in vaccinated monkeys . The numbers correspond to the number of secretory cells forming one spot per million (10 ⁶ ) of peripheral blood mononuclear cells (PBMC). The weeks of analysis are reported at the top of the table.

2.6.2. Analysis of T-cell immune responses by multiparametric flow cytometry in vaccinated monkeys

The results of the multiparametric flow cytometric analyzes confirm those obtained by ELISPOT by revealing that all the animals have developed a response composed of proliferating T cells that are specific for all the antigens expressed by the vaccine vector (Table 4). These responses are heterogeneous between animals and also reveal a first phase of primary response that extends to about 8 weeks PI, followed by a contraction phase (2-4 weeks) and then a re-emergence phase. This longitudinal monitoring of the immune response by multiparametric flow cytometry is continued until the virulent challenge by the test virus SIVmac251 which is carried out at week 52.

Proportion of T cells that proliferate in response to

Ag Antigenic Animals (Ag)

Week Week Week Week

Type

(-D (3-6) (8-20) (22-26)

CD8 + 0 4.2 0.7 3.0

Gag

CD4 + 0.6 0.2 0.4

CD8 + ND 0.7 0.2 0.7

Pol

CD4 + ND 1 .3 0.1 0.1

BX72

CD8 + 0.2 1 .0 0.5 0.6

ca.

CD4 + 0.3 1 .4 0.5 2.3

CD8 + 0.1 0.9 0.3 1 .5

TRN CD4 + 0.1 1 .7 0.4 0.9

Week Week Week Week

Type

(-D (3-6) (8-18) (20-32)

CD8 + 0 3.4 1 .1 3.7

Gag

CD4 + 0.1 1 .3 0.6 2.9

CD8 + ND 1 .1 0.3 1 .1

Pol

CD4 + ND 0.6 0.2 1 .3

BX78

CD8 + 0.2 1 .4 0.4 1 .2

ca.

CD4 + 0.3 0.6 0.6 2.5

CD8 + 0.3 1 .9 0.8 2.1

TRN CD4 + 0.3 1 .2 0.5 2.1

Table 4: Summary of proliferative CD4 + and CD8 + T cell values in response to antigenic stimulations at the time of the primary expansion, contraction and finally reemergence or secondary expansion phases in vaccinated monkeys. The weeks corresponding to each of the phases are indicated. The numbers correspond to the percentages of T cells specific for each of the antigens that proliferate in response to stimulation relative to the total number of T cells.

2.6.3. Analysis of the humoral response in macaques

The detection of anti-SHIV antigen antibodies has been performed by a commercial ELISA kit which makes it possible to detect anti-HIV-1 Env antibodies. Longitudinal examination of the sera collected at each blood collection point showed the presence of anti-Env antibodies from week 20 PI in animal BX80 and from week 8 for animal BX73. The presence of antibodies in the positive sera was confirmed by Western blotting against SHIV proteins showing a strong signal against Gag-p27 protein as well as a signal against glycoproteins gp160 / gp120.

2.6.4. Conclusion

These results clearly demonstrate that a single injection of vaccinia DNA (CAL-SHIV-IN-) makes it possible to induce T cell and humoral immune responses (antibodies). T cell responses are directed against all viral antigens expressed by the vaccine vector. They are persistent and pursue a classic pattern of expansion, contraction and memorization. The presence of central memory and memory effector T cells was confirmed by phenotyping.

2.7. Construction and functional analysis of a new vector expressing the set of HIV-1 antigens: the vector CAL-HIV-IN-

From the genome of the CAL-SHIV _K U2-IN- vector, the SIV gag, pol, vif, vpx and vpr genes were deleted following double digestion with the Nar1 and Sph1 enzymes, and the SIV nef gene. was deleted following partial digestion with the enzyme Nru1 and total digestion with the enzyme Not 1. The remaining fragment was then purified. This fragment, carrying the tat, rev and env genes of the HIV boxed by the LTRs of CAEV and transported by the plasmid pET, was used to introduce the 5kb fragment carrying the gag, pol, vif, vpx and vpr genes of HIV. 1 and the 620 bp fragment carrying the nef gene of HIV-1, and generate the CAL-HIV-IN- vector (FIG. 17). The vector CAL-HIV-IN-, deleted coding sequences of the integrase consists of the sequence SEQ ID NO: 18.

2.7.1. Evaluation of Functionality: Effect of Plasmid DNA on Cells

The DNA of this vector was introduced into GHOST-CXCR4 and HEK-293 T cells by transfection using ExGen and the protocol recommended by the manufacturer. The transfected GHOST-CXR4 cells then became fluorescent attesting to the expression of the viral proteins of HIV-1 by the vaccine vector and more particularly the Tat protein which transactivates the expression of the GFP (Green Fluorescent Protein) gene under the control of the HIV LTR.

The supernatant of HEK-293T cells transfected with the vaccine vector CAL-HIV-IN- was used to inoculate M8166 indicator CD4 + T cells that developed characteristic cytopathic effects of HIV infection. These results provide evidence that the proteins expressed by the vaccine vector have assembled into viral particles allowing the infection of M8166 indicator cells. These cells with cytopathic effects did not produce a virus capable of re-infecting M8166 indicator cells and inducing cytopathic effects. This result indicates the CAL-HIV-IN- is associated with a single cycle of replication in the absence of integration.

To evaluate the viral proteins produced after transfection, the supernatants of HEK-293T cells transfected with the vaccine vector were harvested 24h, 48h and 72h post transfection and then examined for the presence of Gag p24 antigens by ELISA. Measurements of the amounts of this protein are reported in Table 5. demonstrate an increasing accumulation of this protein ranging from 100 ng / ml to 24h up to 135 ng / ml at 72 hours post transfection.

Table 5: Evaluation of secreted H IV-1 Gag p24 protein in the supernatant of HEK 293T cells transfected with the vaccine vector CAL-HIV-IN-. Quantification of the p24 protein accumulated in the supernatant of the HEK 293T cells after 24, 48 and 72 h post-transfection with the CAL-HIV-IN-vector DNA.

2.7.2. Immunization of BALB / C Mice and Characterization of Induced Immune Responses Three groups of BALB / c mice (6 per group) of 6 weeks were used: 2 groups were used for immunization and the third group was a control group. The 2 groups of immunized mice were injected with 100 μg / mouse of CAL-HIV-IN-vector DNA by the intramuscular route. Animals in one group were sacrificed at 2 weeks and the other at 3 weeks post immunization (PI). The control mice were sacrificed at 2 weeks Pl. The spleens of each of the mice were removed, the splenocytes were isolated and then used either for the ELISPOT assay or for the multiparametric flow cytometry analysis as described above. The results of the ELISPOT analysis are reported in Table 6. They demonstrate the presence of specific cells of all the antigens that secrete IFN-λ. The majority of these cells are specific for the Gag and Tat + Rev + Nef antigens. The responses of weeks 2 and 3 post immunization are substantially similar.

Table 6: Summary of the results of the ELISPOT analysis on splenocytes of BABL / c mice immunized with the vaccine vector CAL-HIV-IN- at 2 and 3 weeks post-immunization. The splenocytes isolated from the spleens of mice immunized with 100 μg per intramuscular mouse were examined by the IFN-λ ELISPOT test to evaluate the number of T cells secreting this cytokine in response to the stimulations with the peptide pools (Gag, Env and Tat + Rev + Nef, TRN). Spot number averages for each antigen and for the 6 mice examined after 2 and 3 weeks post-immunization are reported. The results of the flow cytometric analysis (Table 7) demonstrate immune responses in CD4 + and CD8 + T cells specific for all the antigens expressed by the vaccine vector CAL-HIV-IN-.

Table 7: Summary of the results of the multiparametric flow cytometry analysis. The figures correspond to the percentages of T cells specific for each of the antigens that proliferate in response to the antigenic stimulation relative to the total number of T cells.

Claims

1. A nucleic acid comprising a chimeric retroviral genome, wherein said chimeric retroviral genome comprises:

the 5 'and 3' repeated terminal sequences (LTR) of a first retrovirus, said first retrovirus being the arthritis-encephalitis Caprine virus (CAEV), the Maedi Visna virus (VMV), the viral virus; Equine infectious anemia (EIAV), an oncovirus, or spumavirus, and

2. Nucleic acid according to claim 1, characterized in that the chimeric retroviral genome comprises at least three viral genes of said second retrovirus.

3. Nucleic acid according to claim 1 or 2, characterized in that the chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said second retrovirus.

4. Nucleic acid according to claim 1 or 2, characterized in that the chimeric retroviral genome further comprises at least one viral gene of a third retrovirus, said third retrovirus being different from said first and second retrovirus.

5. A nucleic acid according to claim 1, wherein said at least one gene of said second retrovirus or third retrovirus is selected from gag, pol, vif, vpx , rev, vpu and approx.

6. Nucleic acid according to any one of claims 1, 2, 4 and 5, characterized in that the chimeric retroviral genome comprises the genes gag, pol, vif, vpx, vpr, nef of said second retrovirus and tat genes, rev , vpu and env of said third retrovirus.

7. Nucleic acid according to any one of claims 1 to 3, characterized in that said second retrovirus is selected from an oncovirus, a lentivirus or a spumavirus.

8. Nucleic acid according to any one of claims 1, 2, and 4 to 6, characterized in that said second and third retrovirus are each selected from an oncovirus, a lentivirus or a spumavirus.

9. Nucleic acid according to any one of claims 1, 2, and 4 to 8, characterized in that said second and third retrovirus are each a lentivirus.

10. Nucleic acid according to any one of claims 7 to 9, characterized in that said lentivirus is selected from the human immunodeficiency virus type I (HIV-1), HIV-2, the virus of the simian immunodeficiency (SIV), feline immunodeficiency virus (FIV) or equine infectious anemia virus (EIAV).

1 1. Nucleic acid according to any one of claims 1, 2 and 4 to 10, characterized in that said lentiviral chimeric genome comprises gag, pol, vif, vpx, SIV vpr genes and nef, tat, rev, vpu and HIV-1 env.

12. Nucleic acid according to any one of claims 1 to 1 1, characterized in that said lentiviral chimeric genome comprises gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of HIV-1. or the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of HIV-2.

13. Nucleic acid according to any one of claims 1 to 12, characterized in that said pol gene, when present, is a pol gene deleted sequences encoding the integrase (in).

14. Nucleic acid according to claim 13, characterized in that said non-integrative chimeric retroviral genome comprises the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 6.

A recombinant vector comprising a nucleic acid according to any one of claims 1 to 14.

16. Immunogenic or vaccine composition comprising a nucleic acid according to any one of claims 1 to 14, or a vector according to claim 15.

Nucleic acid according to any one of claims 1 to 14, vector according to claim 15, or vaccine composition according to claim 16, for its use for the prevention or treatment of a retrovirus infection.

18. Nucleic acid, vector, or vaccine composition for their use according to claim 17, wherein the retrovirus is an oncovirus, a lentivirus or a spumavirus.

Nucleic acid, vector, or vaccine composition for their use according to claim 17 or 18, wherein the retrovirus is HIV-1, HIV-2 or FIV.