# Patent

## Title
Chimeric non-integrating lentiviral genomes as vaccines against HIV-1

## Abstract

Novel nucleic acids include non-integrative chimeric retroviral genomes including the 5′ and 3′ long terminal repeat sequences (LTRs) of the caprine lentivirus: the Caprine Arthritis Encephalitis Virus (CAEV) or of another retrovirus not integrating human cells and at least one viral gene of another retrovirus. A vector including such a nucleic acid, an immunogenic or vaccinal composition including the vector or the nucleic acid, as well as their use for treating and/or preventing an infection by a retrovirus or a disease induced by a pathogenic agent are also described.

## Background
The object of the present invention is nucleic acids comprising non-integrative chimeric retroviral genomes comprising the repeated terminal sequences (STR, or LTR for Long Terminal Repeat) 5′ and 3′ of the caprine lentivirus: the Caprine Arthritis Encephalitis Virus (CAEV) or of another retrovirus which does not integrate 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 vaccinal composition comprising said vector or said nucleic acid, as well as to their use for treating and/or preventing an infection by a retrovirus or a disease induced by a pathogenic agent.
At present, the development of effective vaccines against retroviral infections is a major public health challenge worldwide. Recently, vaccines have been developed based on the use of vectors having the capability of expressing immunogenic proteins in the vaccinated host. These vaccinal vectors, after amplification in bacteria, are purified and directly injected into the host requiring vaccination. The vector is thus managed by the cells of the host, the immunogenic proteins are expressed and presented to the molecules of the major histocompatibility complex of class I and II, thereby allowing generation of immune responses against these immunogenic proteins. The first vaccination tests by means of retroviral vaccinal vectors gave the possibility of showing that immunization against the Rous sarcoma virus (Chebloune et al., 1991, J Virol, 65, 5374-5380), the New Castle disease virus (Cosset, Bouquet et al., 1991, Virology 185, 862-866) and then the influenza virus (Robinson, Hunt et Webster, 1993, Vaccine, 11(9): 957-960) was possible in chickens.
This vaccinal approach is particularly of interest for controlling the human immunodeficiency virus (HIV). The acquired immunodeficiency syndrome (AIDS) today continues to be a worldwide public health problem with more than 33 million infected individuals, and with more than 2 million deaths and about 3 million new infections per year. Africa is the most affected continent, but the infection is rapidly growing in Asia and in certain Eastern European countries, this phenomenon being certainly due to the lack of means for early detection and to the lack of treatment of the infection. Further, because of limitations of an economical nature, many patients infected by HIV in developing countries do not benefit from any treatment, and therefore contribute to massive dissemination of the infection. The economical impact of AIDS will therefore certainly be very important during the next few years. In Europe, AIDS remains one of the most significant transmissible pathologies with about one million persons living with AIDS and more than 20,000 new infections per year in Western Europe and in Central Europe; and with about 1.5 million persons living with AIDS and more than 200,000 new infections per year in Eastern Europe. The development of a prophylactic vaccine stopping this infection therefore remains a priority.
In spite of many efforts, to this day, there is no secure and satisfactory vaccine providing protection for humans against infection by the HIV or against the pathogenesis induced by this virus. Nevertheless, much research carried out has given the possibility of accumulating precious knowledge in order to understand the failures of the vaccinal strategies used up to now, and of defining the required properties of a vaccine inducing immune responses giving protection against the lentiviruses responsible for AIDS.
The vaccine should notably induce a CD8+ T lymphocyte response, which is associated with controlling the virus during primary infection, and the presence of which has been shown as being indispensable for controlling the viral load in infected non-human primates (Jin et al., 1999, J Exp Med, 189: 991-998). Further, cytotoxic T lymphocytes (CTL) are present in long term non-progressive patients (LTNP) (Rinaldo et al., 1995, J Virol, 69: 5838-5842), or further in subjects exposed to but not infected by the HIV (Makedonas et al., 2002, AIDS, 16: 1595-1602). These elements and other ones, show the importance of such responses in controlling viral replication and/or preventing the disease.
Further, vaccination should induce a response of CD4+ T cells, which are indispensable for stimulating and maintaining response based on anti-HIV CD8+ T lymphocytes (Kalams et al., 1999, J Virol, 73: 6715-6720). CD4+ T cells are also indispensable for setting up and maintaining the response based on antibodies produced by B lymphocytes (BL). It was thus shown that macaques infected by the SIV and depleted in BL did not control their viral load as well as control monkeys (Johnson et al., 2003, J Virol, 77: 375-381). Considering these results and the foregoing results, it seems therefore to be necessary that a vaccine against HIV should stimulate the B and T responses of the immune system.
Among the many tested vaccinal strategies, are found those involving so called attenuated lentiviruses. It was thus shown that the latter gives the possibility of reproducibly inducing the best protection against homologous and heterologous test viruses (Yankee et al., 2009, Virology, 383: 103-111; 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 genome of the host and of the recurrent infection probability related to proviral latencies, these viruses are pathogenic in certain 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 ethical and safety reasons, these attenuated lentiviruses cannot therefore be used as such in humans.
DNA vaccination based on viral vectors, as for it, has never been associated with development of pathologies, either in humans, or in animals, and consequently is more safe. However, tested in monkeys, these vectors prove to be incapable of protecting the animals against an experimental infection (Liu et al., 2006, Virology, 351: 444-454; Singh et al., 2005, J Virol, 79: 3419-3428).
Therefore, there exists the need for novel vaccinating vectors allowing expression of the lentiviral antigens at higher levels both in quantity and in quality, with the purpose of inducing protective responses against pathogenic viruses.
Previously, the inventors have described infectious viral genomes comprising a complete viral genome including the RTLs of the 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 were used for studying the mechanisms of pathogenesis induced by highly pathogenic retroviruses of humans and monkeys.
The inventors have discovered that the use of Repeated Terminal Sequences (RTS, or LTR for Long Terminal Repeat) of the Caprine Arthritis Encephalitis Virus (CAEV) gave the possibility of improving the expression of vaccinating retroviral genomes and the induction of protective responses against pathogenic retroviruses, while avoiding their integration into the host cells. The inventors in particular demonstrated that the Long Terminal Repeat Sequences (LTRs) of the Caprine Arthritis Encephalitis Virus (CAEV) allowed constitutive expression of the genes associated with them and were not dependent on the viral tat gene of the CAEV, more particularly on the protein of the viral tat gene of the CAEV, for expressing the genes of a viral genome to which they are merged, thus allowing strong expression of viral antigens. The inventors thus developed chimeric genomes, between the lentiviruses of primates of the SIV and HIV type (HIV for Human Immunodeficiency Virus) and the CAEV, which have the properties of not being integrative and non-replicative, while being capable of carrying out a replication cycle for expressing all the antigens of the HIV and of the SIV present in the genomes. The inventors demonstrated that transfection of these genomes in primate cells (HEK293) allows expression of all the proteins of the genes present and that these proteins are assembled into viral particles capable of carrying out a single infection cycle (i.e. a pseudo-cycle) in target cells, without integrating the viral genome into these target cells. The immunization of NOD/SCID mice, humanized with human mononuclear cells demonstrated the presence of strong specific humoral and cellular immune responses against viral antigens.
Definitions
By nucleic acid, is meant the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or of deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; “DNA molecules”) in a monoquaternary form or in the form of a bi-quaternary helix. Bi-quaternary helices DNA-DNA, DNA-RNA and RNA-RNA are possible. The term of nucleic acid, and in particular of DNA or RNA molecule, only refers to the primary or secondary structure of the molecule, and is by no means limited to particular tertiary forms. Thus, this term comprises bi-quaternary DNA which is found, inter alia, in linear or circular DNA molecules (for example, restriction fragments), viruses, plasmids and chromosomes. When the structure of particular bi-quaternary DNA molecules is mentioned, the sequences may be described here according to the normal convention which only gives the sequence in the 5′ to 3′ direction along the non-transcribed strand of the DNA (i.e. the strand having a homologous sequence to the mRNA).
Within the context of the invention, a nucleic acid comprising a non-integrative chimeric retroviral genome refers to a nucleic acid which comprises the nucleic acid sequences acting in the cis position of at least two retroviruses, said nucleic acid not being capable of integrating the genome of a host cell. The nucleic acid includes the Long Terminal Repeat Sequences (LTR) in 5′ and 3′ of a first retrovirus, and at least one viral gene of a second retrovirus.
By “retrovirus”, is meant a virus for which the genome consists of an RNA molecule and which comprises a reverse transcriptase, i.e., a member of the family of Retroviridae. The retroviruses are divided into three kinds: oncoviruses, lentiviruses and spumaviruses. Oncoviruses notably consist of the following species: the murine leukemia virus (MLV), the avian leukosis virus (ALV), the Rous sarcoma virus (RSV for Rous Sarcoma Virus), or the simian Mason-Pfizer virus. Lentiviruses consist of the following species: the human immunodeficiency virus of type 1 (HIV-1), the human immunodeficiency virus of type 2 (HIV-2), the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV), the bovine immunodeficiency virus (BIV), the sheep Visna Maedi virus (VMV), the Caprine Arthritis Encephalitis Virus (CAEV) or the Equine Infectious Anaemia Virus (EIAV). The spumavirus may be HFV. When the retrovirus is HIV-1, it may be of any serogroup, for example of serogroup M (serotype A-D, F-H, J, K), serogroup O, N or P. When the retrovirus is HIV-2, it may be of any serogroup, for example of serogroup A or B.
By viral gene, is meant a gene present in the retroviral genome. Within the context of the invention, the viral gene may be gag, pol, vif, vpx, vpr, nef, tat, rev, vpu or env gene.
The gene gag meaning group specific antigen codes for the precursor polyprotein gag which is cleaved for giving the fundamental structural proteins of the retroviruses, which are the capsid proteins, proteins of the nucleocapsid, and the proteins of the matrix. For example, the protein gag of the HIV is the precursor of the capsid protein p24, of the proteins of the p6 and p7 nucleocapsid, and of the protein of matrix p17. As a non-limiting example, the gene gag is the gene gag of the HIV-1 (NCBI gene ID No. 155030, updated on Aug. 20, 2011), of the HIV-2 (NCBI gene ID No. 14900001, updated on Aug. 27, 2011), of the SIV (NCBI gene ID No. 956108, updated on Aug. 27, 2011) or of the FIV (NCBI gene ID No. 1489988, updated on Aug. 20, 2011).
The gene pol codes for a reverse transcriptase, an integrase and a protease. As a non-limiting example, the gene pol is the gene pol of the HIV-1 (NCBI gene ID No. 155348, updated on Aug. 27, 2011), of the HIV-2 (NCBI gene ID No. 1490001, updated on Aug. 27, 2011), of the FIV (NCBI gene ID No. 1489989, updated on Aug. 27, 2011) or of the SIV (NCBI gene ID No. 956107, updated on Aug. 20, 2011).
The gene vif or viral infectivity factor codes for a protein required for producing infectious virions. As a non-limiting example, the gene vif is the gene vif of HIV-1 (NCBI gene ID No. 155459, updated on Aug. 7, 2011), of the HIV-2 (NCBI gene ID No. 1724712, updated on Jan. 21, 2010), of FIV (NCBI gene ID No. 1724709, updated on Feb. 7, 2010) or of the SIV (NCBI gene ID No. 1490005, updated on Jan. 21, 2010).
The gene vpr codes for the viral protein R which plays an important role in the stopping of the cell cycle in phase G2, and in the regulation of the transport of the pre-integration complex from the cytoplasm to the nucleus, the viral replication. As a non-limiting example, the gene vpr is the gene vpr of HIV-1 (NCBI gene ID No. 155807, updated on Aug. 7, 2011), of HIV-2 (NCBI gene ID No. 1724718, updated on Jan. 21, 2010) or of SIV (NCBI gene ID No. 956112, updated on Jan. 15, 2011).
The gene vpx code for the viral protein X is related to the gene vpr. As a non-limiting example, the gene vpx is the gene vpx of HIV-2 (NCBI gene ID No. 1724714, updated on Mar. 19, 2011) or of SIV (NCBI gene ID No. 1490006, updated on Jul. 16, 2011).
The gene nef codes for the myristoylated protein of 27 to 25 kDa, called a Negative Regulation Factor, which plays a key role in the depletion of CD4 lymphocytes in vivo. As a non-limiting example, the gene nef is the gene nef of HIV-1 (NCBI gene ID No. 156110, updated on Aug. 7, 2011), of HIV-2 (NCBI gene ID No. 1724715, updated on Mar. 19, 2011) or of SIV (NCBI gene ID No. 1490008, updated on Jul. 2, 2011).
The gene tat codes for a protein of 86 to 101 amino acids, called a Trans-Activator of Transcription, which increases the transcription rate of the retroviral genome. As a non-limiting example, the gene tat is the gene tat of HIV-1 (NCBI gene ID No. 155871, updated on Aug. 20, 2011), of HIV-2 (NCBI gene ID No. 1724713, updated on Feb. 7, 2010) or of SIV (NCBI gene ID No. 956113, updated on Feb. 7, 2010).
The gene rev codes for a protein called Regulator of Virion Expression which allows export of the viral RNA from the nucleus to the cytoplasm. As a non-limiting example, the gene rev is the gene rev of HIV-1 (NCBI gene ID No. 155908, updated on Aug. 7, 2011), of HIV-2 (NCBI gene ID No. 1724716, updated on May 21, 2011) or of SIV (NCBI gene ID No. 1490003, updated on Jan. 15, 2011).
The gene vpu codes for a protein called a Viral Protein U which is involved in viral budding and improvement in the release of virions. As a non-limiting example, the gene vpu is the gene vpu of HIV-1 (NCBI gene ID No. 155945, updated on Aug. 7, 2011) or of SIV (NCBI gene ID No. 2828723, updated on Jan. 21, 2010).
The gene env codes for the precursor protein gp160 which is ripened and cleaved in order to give the proteins of the envelope gp120 and gp41. As a non-limiting example, the gene env is the gene env of HIV-1 (NCBI gene ID No. 155971, updated on Aug. 7, 2011), of HIV-2 (NCBI gene ID No. 1724717, updated on Jun. 18, 2011), of FIV (NCBI gene ID No. 1489987, updated on Jun. 18, 2011) or of SIV (NCBI gene ID No. 1490007, updated on Jun. 18, 2011).
In the sense of the present application, the term of comprises or comprising refers according to a particular mode to consist in or consisting in.
Nucleic Acid
The inventors have demonstrated that Long Terminal Repeat Sequences (LTR) of the Caprine Arthritis Encephalitis Virus (CAEV) allowed constitutive expression of the genes associated with them and were not dependent on the viral gene tat for expressing the genes of a viral genome to which they are merged, thus allowing strong expression of viral antigens. Further, the inventors showed that the sole presence of these LTRs prevented integration of a heterologous retroviral genome to which they are merged.
The invention therefore relates to a nucleic acid comprising a non-integrative chimeric retroviral genome, in which said chimeric retroviral genome comprises:

    
    
        Long Terminal Repeat Sequences (LTR) in 5′ and in 3′ of a first retrovirus, said first retrovirus being a lentivirus, such as the Caprine Arthritis Encephalitis Virus (CAEV), the ovine Visna Maedi virus (VMV), the Equine Infectious Anaemia Virus (EIAV), or an oncovirus or a spumavirus, and
        at least one viral gene of a second retrovirus, said second retrovirus not being the first retrovirus.
    
    


The LTR sequences used preferably stem from a retrovirus which does not integrate 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 may be integrated. For example, when the first retrovirus is CAEV, said host or patient is not a caprine but may be a human, a monkey, a cat, or a horse, or when the first retrovirus is EIAV, said host or patient is not a horse but may be a human, a monkey, a cat, or an ovine, or when the first retrovirus is VMV, said host or patient is not an ovine but may be a human, a monkey, a cat, or a horse.
In a particularly preferred embodiment, the first retrovirus is the Caprine Arthritis Encephalitis Virus or CAEV. The CAEV is a retrovirus of the lentivirus type of goats which is related to the human immunodeficiency virus (HIV), but does not cause any pathology of the AIDS type in its host.
By Long Terminal Repeated Sequences (LTR), is meant a sequence allowing control of the transcription, i.e. comprising an enhancer, a promoter, one or several signals for initiating the transcription, one or several signals for ending the transcription, one or several signals for poly-adenylation. Within the context of the invention, the LTRs of CAEV comprise an enhancer, a promoter and a signal for initiating transcription as well as a signal for ending the transcription and a poly-adenylation signal. Preferably, the LTRs in 5′ and in 3′ of the CAEV are identical and comprise or consist in the sequence SEQ ID NO.: 3.
In other embodiments, the LTRs of the Visna Maedi Virus (VMV) or those of the lentivirus of equidae EIAV are used. The LTR in 5′ of VMV comprises or consists in the sequence found in position 1 to 161 of the Reference Sequence NCBI NC_001452.1 (updated on Dec. 8, 2008). The LTR in 3′ of VMV comprises or consists in the sequence found in position 9106 to 9202 of the Reference Sequence NCBI NC_001452.1 (updated on Dec. 8, 2008). The LTR in 5′ of EIAV comprises of consists in the sequence found in position 61 to 381 of the Reference Sequence NCBI NC_001450 (updated on Mar. 11, 2010). The LTR in 3′ of EIAV comprises or consists in the sequence found in position 7269 to 8289 of the Reference Sequence NCBI NC_001450 (updated on Mar. 11, 2010).
In still other embodiments, the LTRs of an oncovirus, such as the murine leukemia virus (MLV), the avian leukemia virus (ALV), the Rous sarcoma virus (RSV), or the simian Mason-Pfizer virus, or the LTRs of a spumavirus, such as HFV are used. The LTR in 5′ of the MLV comprises or consists in the sequence found in position 1 to 210 of the Reference Sequence NCBI NC_001702.1 (updated on Feb. 5, 2011). The LTR in 3′ of the MLV comprises or consists in the sequence found in position 5735 to 8135 of the Reference Sequence NCBI NC_001702.1 (updated on Feb. 5, 2011). The LTR in 5′ of the ALV comprises or consists in the sequence found in position 1 to 594 of the Reference Sequence NCBI NC_015116.1 (updated on Apr. 18, 2011). The LTR in 3′ of the ALV comprises or consists of the sequence found in position 5338 to 7489 of the Reference Sequence NCBI NC_015116.1 (updated on Apr. 18, 2011). The LTR in 5′ of RSV comprises or consists of the sequence found in position 22 to 102 of the Reference Sequence NCBI NC_001407.1 (updated on Dec. 8, 2008). The LTR in 3′ of the RSV comprises or consists of the sequence found in position 9058 to 9292 of the Reference Sequence NCBI NC_001407.1 (updated on Dec. 8, 2008). The LTR in 5′ of the simian Mason-Pfizer virus comprises or consists of the sequence found in position 26 to 123 of the Reference Sequence NCBI NC_001550.1 (updated on Dec. 8, 2008). The LTR in 3′ of the simian Mason-Pfizer virus comprises or consists of the sequence found in position 7573 to 7811 of the Reference Sequence NCBI NC_001550.1 (updated on Dec. 8, 2008). The LTR in 5′ of the HFV comprises or consists of the sequence found in position 1 to 1760 of the Reference Sequence NCBI NC_001364.1 (updated on Apr. 22, 2009). The LTR in 3′ of the HFV comprises or consists of the sequence found in position 11487 to 13246 of the Reference Sequence NCBI NC_001364.1 (updated on Apr. 22, 2009).
The inventors have shown that the Long Terminal Repeat Sequences (LTR) of the Caprine Arthritis Encephalitis Virus (CAEV) were not dependent on the viral gene tat for expressing the genes of a viral genome to which they are merged, advantageously, the nucleic acid according to the invention does not contain the tat gene of said first retrovirus.
Within the context of the invention, the second retrovirus is different from the first retrovirus. It may be an oncovirus, a lentivirus or a spumavirus. Thus, for example, when the LTRs of the CAEV are used, the second retrovirus is not the CAEV. Preferably, the second retrovirus is an oncovirus, such as the murine leukemia virus (MLV), the avian leukosis virus (ALV), the Rous sarcoma virus (RSV), or the simian Mason-Pfizer virus, a lentivirus, such as the human immunodeficiency virus of type 1 (HIV-1), the human immunodeficiency virus of type 2 (HIV-2), the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV) or the equine infectious anaemia virus (EIAV), or a spumavirus, such as HFV. More preferably, the second retrovirus is HIV-1, HIV-2, SIV or FIV.
Preferably, at least one viral gene of said second retrovirus is selected from the 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 (for example the genes gag, pol, vif, or the genes gag, pol, env), four, five, six, seven, eight, nine, ten viral genes of said second retrovirus. Advantageously, said chimeric retroviral genome comprises the gene tat of said second retrovirus. Still more preferably, said chimeric retroviral genome comprises the set of genes gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env 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 env genes of the SIV, HIV-1, HIV-2 or FIV. In a still preferred aspect, said chimeric retroviral genome comprises or consists of the sequence of the retroviral genome of SIV (SEQ ID NO.: 4), of the retroviral genome of HIV-1 (SEQ ID NO: 2), of the retroviral genome of HIV-2 (SEQ ID NO: 5), or the retroviral genome of FIV (SEQ ID NO: 6).
The chimeric retroviral genomes of the SIV, HIV-1, HIV-2 and FIV are schematically illustrated in FIGS. 1 to 4, respectively.
In a particular embodiment, said chimeric retroviral genome further comprises at least one viral gene of a third retrovirus, said third retrovirus not being the first retrovirus, i.e. being different from said first retrovirus. Thus, for example, when the LTRs of the CAEV are used, said third retrovirus is not the CAEV. Said third retrovirus may be selected 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 retrovirus and third retrovirus may be or 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 respectively be (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.
Preferentially, 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 each are a lentivirus, and preferably, said lentivirus is selected from HIV-1, HIV-2, SIV, FIV or EIAV. Still 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 further when said second retrovirus is HIV-1 of serogroup M, said third retrovirus is HIV-1 of serogroup 0, or further when said second retrovirus is HIV-1 of serogroup M and of serotype 1, said third retrovirus is HIV-1 of serogroup M and of serotype B.
Preferably, said at least one viral gene of said third retrovirus is selected from the 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, advantageously including the gene tat.
Still more preferably, said chimeric retroviral genome further comprises the set of genes gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env of said third retrovirus, i.e. 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, tat, rev, vpu and env genes of said third retrovirus.
Said chimeric retroviral 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 of 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 one viral gene of said third retrovirus. As non-limiting 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 vif, vpx, vpr, nef, tat, rev, vpu and env genes 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, vif, vpx, vpr of said second retrovirus and the nef, tat, rev, vpu and env genes 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 gag, pol, vif, vpx, vpr, nef, tat genes of said second retrovirus and the rev, vpu and env genes 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 the SIV and the nef, tat, rev, vpu and env genes of the HIV-1, or conversely the gag, pol, vif, vpx and vpr genes of the HIV-1 and the nef, tat, rev, vpu and env genes of the SIV. In another particularly preferred aspect, said chimeric retroviral genome comprises the gag, pol, vif, vpx and vpr genes of the HIV-1 and the nef, tat, rev, vpu and env genes of the HIV-2, or conversely the gag, pol, vif, vpx and vpr genes of the HIV-2 and the nef, tat, rev, vpu and env genes of the HIV-1. In a still more preferred aspect, said chimeric retroviral genome comprises or consists in sequence SEQ ID NO: 7 (a schematic representation of this chimeric retroviral genome is found in FIG. 5).
In a particular embodiment, when the pol gene is present in the chimeric retroviral genome, said pol gene is a deleted pol gene from the sequence coding for the integrase (in). Preferably, said pol gene is deleted from the sequence SEQ ID NO: 8 (sequence of the integrase of SIV), SEQ ID NO: 9 (sequence of the integrase of HIV-1), SEQ ID NO: 10 (sequence of the integrase of HIV-2), or SEQ ID NO: 11 (sequence of integrase of FIV).
Thus, in a particularly preferred embodiment, said retroviral genome comprises or consists in the sequences 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 in the sequences SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are schematically illustrated in FIGS. 6 to 10, respectively.
The invention also relates to a vector comprising a nucleic acid according to invention.
The term of vector designates an extrachromosomal element through which a DNA or RNA sequence (i.e. a foreign gene) may be introduced into a host cell, so as to transform the host and to allow expression (i.e. transcription and translation) of the introduced sequence. The extrachromosomal element may be a self-replicating sequence, a phage sequence or a nucleotide sequence, a single or dual strand 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. A common means for inserting a DNA fragment into another DNA segment involves the use of enzymes, called restriction enzymes, which cleave the DNA at specific sites (specific groups of nucleotides), called restriction sites. Generally, the foreign DNA is inserted at one or several restriction sties of the DNA vector, and is then transported by the vector into a host cell with a DNA of the transmissible agent. A DNA segment or sequence comprising an added or inserted DNA, such as the vector, may also be called a DNA construct. A common type of vector is a plasmid, which generally is an autonomous dual strand DNA molecule, generally of bacterial origin, which may easily accept an additional (foreign) DNA and which may easily be introduced into a suitable host cell. A large number of vectors, including plasmids, have been described for the replication and/or expression in different eukaryotic and prokaryotic hosts. Within the context of the invention, the vector includes a selection marker, such as a gene for resistance to an antibiotic, or a nucleic acid according to the invention. Preferably, the resistance gene is a gene for resistance to ampicillin or kanamycin.
The nucleic acids and/or the vector according to the invention may be used for transforming or transfecting a cell or a host organism, i.e. for expressing the chimeric retroviral genome according to the invention.
The term of host cell refers to any cell of any organism which is selected, modified, transformed, transfected, transduced, cultivated, or used or manipulated in any way, for producing a substance by the cell, for example for the expression of a gene, of a DNA sequence, of a protein, of a virion by the cell. Within the context of the invention, the host cell is a mammal cell. Suitable host cells include, without being limited thereto, HEK293 cells, human CD4+ T lymphocyte lines CEMx174 and M8166, human CD4+ T lymphocytes, human CD8+ T lymphocytes, mononuclear cells of human blood.
The transformation of the cell or of the host organism by the nucleic acid and/or the vector according to the invention may be achieved according to standard techniques known to one skilled in the art, such as for example by transfection, electroporation, microinjection, transduction, merging of cells, DEAE-Dextran, precipitation with calcium phosphate, or use of a gene pistol, 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: 14621-14624; Hartmut et al., Canadian patent application No. 2,012,311, published on Mar. 15, 1990).
Immunogenic or Vaccinal Composition and its Uses
The nucleic acid or the vector according to the invention may be used with an immunogenic or vaccinal purpose.
Thus, the invention also relates to an immunogenic or vaccinal composition comprising a nucleic acid or a vector according to the invention.
Within the context of the present application, the term of vaccinal relates to prophylactic or therapeutic vaccination.
By immunogenic or vaccinal composition, is meant a composition giving the possibility of inducing an immune response against a retrovirus as defined earlier. By immune response is meant a response involving T lymphocytes, for example CD4+ and CD8+ T lymphocytes, and B lymphocytes.
According to the embodiment, the immunogenic or vaccinal 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 vaccinal composition according to the invention is multivalent, i.e. it allows an immune response against several retroviruses, for example against HIV-1 and HIV-2 or several pathogenic agents, for example against HIV-1 and HCV (Hepatitis C Virus). In this case, the vaccinating vector expresses the antigens of either pathogenic agent.
According to another embodiment, the immunogenic or vaccinal composition according to the invention is polyvalent. Such an immunogenic or vaccinal composition may be obtained by combining several monovalent immunogenic or vaccinal compositions according to the invention. The immunogenic or vaccinal composition may further comprise at least one other vaccine, i.e. an attenuated live virus, an inactivated virus or a viral sub-unit, against another virus, such as a sexually transmissible virus, such as for example the hepatitis B virus, the hepatitis C virus or the papillomavirus.
In a preferred embodiment, the immunogenic or vaccinal composition according to the invention comprises a pharmaceutically acceptable carrier.
A pharmaceutically acceptable carrier refers to any carrier in which the immunogenic or vaccinal composition according to the invention may be formulated. This includes a saline solution such as a saline phosphate buffer. Generally, a diluent or a carrier is selected according to the administration method and route, and according to standard pharmaceutical practices. A pharmaceutically acceptable carrier includes, without any limitation, iron exchangers, aluminium, aluminium stearate, lecithin, systems for delivering self-emulsifying drugs such as D-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used as a pharmaceutical dosage form such as Tweens or other polymeric delivery matrices, proteins of serum such as human albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated fatty acid glycerides of plants, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers and wool fat. 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 improving delivery of the compositions according to the invention.
The compositions according to the invention may further contain an adjuvant. Any pharmaceutically acceptable adjuvant or mixture of adjuvants conventionally used in the field of vaccines may be used for this purpose. As examples of suitable adjuvants, mention may be made of aluminium salts such as aluminium hydroxide or aluminium phosphate and DC-Chol. Any pharmaceutically acceptable adjuvant or mixture of adjuvants conventionally used in the field of vaccines may be used for this purpose. As an example of a suitable adjuvant, mention may be made of aluminium salts such as aluminium hydroxide or aluminium phosphate and DC-Chol.
The compositions according to the invention may contain adjuvant genes, i.e. genes which express proteins which will play the role of adjuvants by increasing the immunogenicity of the expressed viral proteins. For example, the genes which code for 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 vaccinel plasmid or co-injected as separate expression plasmids.
Any method of administration known to one skilled in the art may be used. In particular, the nucleic acid, the vector, the immunogenic or vaccinel composition according to the invention may be administered orally, by inhalation, or via a parenteral route (in particular by intradermal, subcutaneous, intravenous, intramedullar or intramuscular injection). When the parenteral route is used, the nucleic acid, the vector, the immunogenic or vaccinel composition according to the invention may be in the form of injectable solutions and suspensions, packaged in ampoules or in flasks. The forms for parenteral delivery are generally obtained by mixing the nucleic acid, the vector, the immunogenic or vaccinal composition according to the invention with buffers, emulsifiers, stabilisers, preservatives, solubilizing agents. According to known techniques, these mixtures may then be sterilized and packaged in the forms of intradermal, subcutaneous, intravenous, intramedullar or intramuscular injections. One skilled in the art may use buffers based on organic phosphate salts as a buffer. Examples of emulsifiers include methylcellulose, acacia, sodium carboxymethylcellulose. Examples of stabilisers include sodium sulfite, sodium metasulfite, and examples of preservatives include sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol. The nucleic acid, vector, the immunogenic or vaccinal composition may also be in freeze-dried form.
The vaccinal DNA solution may be directly injected or else administered by electroporation in vivo by using a commercial electroporator or else encapsulated in liposomes or nanoparticles or by using any in vivo transfection method which allows better introduction of the vaccinal DNA into the cells of the vaccinated host.
In one other aspect, the invention relates to a nucleic acid according to the invention, a vector according to the invention and/or an immunogenic or vaccinal composition according to the invention for use in preventing and/or treating an infection by a retrovirus.
By prevent or prevention, is meant the inhibition of a retroviral infection, i.e. preventing the retrovirus from causing an infection, or preventing the propagation of the retrovirus inside an infected subject or from one subject to another.
By treat or treatment, is meant the limitation of the severity of the disease, the prevention of recurrent infections, i.e. limiting the re-activation of latent or persistent infections, and finding a remedy to the symptoms of the infections by a retrovirus. The retrovirus is as defined earlier and preferably the retrovirus is HIV-1, HIV-2 or FIV.
The term of patient or subject or host refers to a human or non-human mammal or a bird. Preferentially, the patient is a primate, a murine (mouse), a feline (cat), a canine (dog), a member of the equidae (a horse), a bird, a human, including women, men, adults and children.
The present invention also relates to a method for vaccinating or treating a subject in need thereof comprising the administration of a prophylactically or therapeutically effective amount of a nucleic acid according to the invention, or of a vector according to the invention, or of an immunogenic or vaccinal composition according to the invention.
A prophylactically or therapeutically effective amount refers to an amount of nucleic acid, of a vector, an immunogenic or vaccinal composition capable of imparting a therapeutic or prophylactic effect on the treated subject. 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 feels an effect). An effective amount may vary from about 0.01 to 5,000 μg/kg, alternatively from about 0.1 to a 1,000 μg/kg, alternatively from about 1 to 500 μg/kg. The effective amounts will also vary according to the administration route, either the use or not of an in vivo transfection method, the size and weight of the subject, as well as according to the possibility of co-use with other agents.
Within the context of the invention, the administration mode of the nucleic acid according to the invention, or of the vector according to the invention, or of the immunogenic or vaccinal composition according to the invention may be achieved via an intravenous, subcutaneous, intradermal, intramedullar, or intramuscular route.
The invention will now be explained in more detail with the following examples, without limiting the scope thereof.



DESCRIPTION OF THE FIGURES
FIG. 1: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 2.
FIG. 2: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 4.
FIG. 3: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 5.
FIG. 4: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 6.
FIG. 5: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 7.
FIG. 6: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 1.
FIG. 7: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 12.
FIG. 8: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 13.
FIG. 9: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 14.
FIG. 10: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 15.
FIG. 11: Schematic illustration of the construction of the genome of CAEV, of the plasmid pSHIVKU2, pCA-LTR-SHIVKU2IN- and of pΔ4SHIVKU2.
FIG. 12: Evaluation of the number of T lymphocytes secreting IFN-γ of Balb/c mice. Spleen cells of control BALB/c immunocompetent mice and immunized with pΔ4SHIVKU2 and pCA-LTR-SHIVKU2IN- and stimulated by the Gag, Env peptides and the peptide Tat+Rev+Nef pool. The number of spots was calculated for 1 million PBMCs.
FIG. 13: Evaluation of the number of human T Lymphocytes secreting IFN-γ in immunized NOD/SCID-hu mice. Spleen cells of immunodeficient mice reconstructed by mononuclear cells of human blood and immunized with pΔ4SHIVKU2 or pCA-LTR-SHIVKU2IN- or pSHIVKU2 are stimulated with Gag, Env peptides and with the peptide pool Tat+Rev+Nef. The number of spots was calculated for 1 million PBMCs and normalized to 20%.
FIG. 14: Illustration of the preparation of the vector CA-LTR-SHIVKU2-IN-.
FIG. 15: Schematic illustration of the vector CA-LTR-SHIVKU2.
FIG. 16: Schematic illustration of the vector CA-LTR-SHIVKU2-IN-.
FIG. 17: Illustration of the preparation of the vector CAL-HIV-IN-.
FIG. 18: Schematic illustration of the vector CAL-HIV-IN-.



DESCRIPTION OF THE SEQUENCES


    
    
        SEQ ID NO: 1 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the SAEV and the genome of SHIV deleted from the sequences coding for the integrase.
        SEQ ID NO: 2 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of the HIV-1.
        SEQ ID NO: 3 Represents the sequence of the LTR of the CAEV.
        SEQ ID NO: 4 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of SIV.
        SEQ ID NO: 5 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of HIV-2.
        SEQ ID NO: 6 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of the FIV.
        SEQ ID NO: 7 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of SHIV.
        SEQ ID NO: 8 Represents the sequence of the integrase of SIV.
        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 integrase of FIV.
        SEQ ID NO: 12 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of HIV-1 deleted from the sequences coding for the integrase.
        SEQ ID NO: 13 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of HIV-2 deleted from the sequences coding for the integrase.
        SEQ ID NO: 14 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and genome of FIV deleted from the sequences coding for the integrase.
        SEQ ID NO: 15 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of SIV deleted from the sequences coding for the integrase.
        SEQ ID NO: 16 Represents the sequence of the vector pCA-LTR-SHIVKU2.
        SEQ ID NO: 17 Represents the sequence of the vector pCA-LTR-SHIVKU2-IN-.
        SEQ ID NO: 18 Represents the sequence of the vector CAL-HIV-IN-.
    
    


EXAMPLES
1. Material and Methods
1.1. The Vaccinating Vectors (FIG. 1)
1.1.1. The Vectors pCA-LTR-SHIVKU2 and pCA-LTR-SHIVKU2-IN-
The vector CA-LTR-SHIVKU2 contains the genome of the Simian and Human Immunodeficiency Virus (SHIV) deleted from the LTRs of SIV and replaced with the LTRs of CAEV. The SHIV contains a chimeric genome consisting of the one of SIV-mac239 in which the tat, env and rev genes of the SIV were deleted and replaced with the vpu, tat, env and rev genes of the HIV-1. The vector therefore bears the vpr, vpx, gag, pol, vif and nef genes of SIV and the tat, rev, vpu and env genes of HIV-1 under the transcriptional control of the LTRs in 5′ and 3′ of the CAEV. The pol gene was deleted from the sequences coding for the integrase (in). The vaccinating vector pCA-LTR-SHIVKU2 non-deleted from the sequences coding for the integrase consists in the sequence SEQ ID NO: 16 (FIG. 15). The vector pCA-LTR-SHIVKU2-IN-, deleted from the sequences coding for the integrase consists in the SEQ ID NO: 17 (FIG. 16).
The construction of a vector CA-LTR-SHIVKU2-IN- was achieved in the following way (FIG. 14). The vector SHIV-KU2 was digested with EcoR1 and Nar1, and then the LTR fragment of 0.8 kb was removed. The CAEV-pBSCA vector was then digested with EcoR1 and Nar1 and the fragment LTR of 0.5 kb was purified. Both fragments were then subject to ligation. The vector SHIV-1 LTRCA was then digested with Stu1 and Ava1 and the LTR fragment of 0.8 kb was removed. The LTR in 3′ of the CAEV was amplified with timers Stu1 and Ava1, the PCR products were digested by Stu1 and Ava1 and the LTR fragment of 0.5 kb was purified. Both fragments were subject to ligation for generating the CAL-SHIVKU2. Finally a digestion with Kpn1 and Acc1 in the pol gene was achieved in order to remove 314 bp of the gene of the integrase of SHIV for generating the CAL-SHIVKU2-IN-.
1.1.2. Vectors pSHIVKU2 and Δ4SHIVKU2 
The plasmids pSHIVKU2 and pΔ4SHIVKU2 are plasmids used as controls. Their constructions have been described in many publications (Liu Z Q et al., 2006, Ramakrisna Hegde et al., 2005).
1.2 Production of Vaccinal DNA
1.2.1. Bacterial Culture
E. coli K12 (JM109) bacteria containing the plasmid are put in a pre-culture in 5 ml of BL medium containing 0.05 mg/ml of kanamycin and then incubated for one night at 30° C. with stirring at 150 rpm. From the pre-culture, the bacterial suspension was diluted to 1:100,000 in the BL medium, and then 50 μl of the dilution are spread over the surface of the agar/BL/Kanamycin contained in a Petri dish which is then incubated at 32° C. for one night. The isolated colonies developed on the agar of the Petri dish are sown into 5 ml of liquid medium BL containing 0.05 mg/ml of kanamycin and cultivated with stirring at 150 rpm at 30° C. for one night. A fraction (1 ml) of the culture is used for rapid extraction of the DNA by means of the Mini-prep kit of Macherey-Nagel or Qiagen, according to the recommended procedure, and the extracted DNA is then separated on a 1% agarose gel for checking its quality. The bacteria corresponding to the DNA estimated to be satisfactory are used for sowing the 1 L cultures which are cultivated under the same conditions as earlier, for the isolation of DNA in a maxi-preparation.
1.2.2. Maxi-Preparation: Plasmid Extraction
The bacteria cultivated with stirring (150 rpm) at 30° C. for one night are harvested as a sediment by centrifugation (4000 g, 4° C., 15 min) and the sediment is re-suspended in 8 ml of re-suspension buffer (Tris-HCl 50 mM pH8, EDTA 10 mM). The cells are then lyzed by adding 8 ml of alkaline lysis buffer (NaOH 200 mM, 1% SDS) in order to release the plasmid DNA. The lyzate is neutralized by adding 8 ml of neutralization buffer (potassium acetate 3M pH 5.5). The mixture is then incubated for 5 min in ice and then centrifuged for 15 minutes at 15,000 g at 4° C. The solution containing the DNA is transferred into a column equilibrated beforehand, allowing retention of the plasmid DNA. The column is washed three times with the washing buffer and then the DNA is eluted and precipitated with isopropanol. The precipitated DNA sediment 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° C. at 15,000 g in order to remove the excess impurities and salts and then the sediment is dried and re-suspended in a suitable volume of ultra-pure water.
The concentration of the DNA solution was then determined by spectrophotometry at a wavelength λ equal to 260 nm and the quality of the plasmid is then checked by electrophoretic migration on a 1% agarose gel. The size of the plasmid and the integrity of the plasmid are checked on an agarose gel after digestion by restriction enzymes Bam H1 and Eco R1 for example.
1.2.3. Checking the Plasmid pCA-LTR-SHIVKU2-IN- by Enzymatic Digestion
An aliquot fraction of 0.5 μg of the plasmid is subjected to digestion with 2 units of enzymes EcoR1, BamH1 or Sph1 for 60 minutes at 37° C. in a suitable 1× buffer for each enzyme, and in a final volume of 20 μl. The profile of the digestion is checked by electrophoretic migration in a 1% agarose gel with a TAE 1× buffer and revealed with ethidium bromide (ETB) and observation of the gel under UVs.
1.3 Cell Cultures and Treatments
1.3.1. Cell Models and Cultivation Conditions
The cell lines were obtained from the National Institute of Health AIDS Research and Reference Reagent Program in the United States. The cells are cryo-preserved in 10% of dimethylsulfoxide (DMSO), at −170° C. in liquid nitrogen. They are defrosted and then cultivated in cultivation flasks.
HEK293T (immortalized Human Embryonic Kidney 293) cells are a permanent line of human embryonic kidney cells. They are used because they are very easy to transfect, with very high transfection efficiencies which may attain 100%. The lentiviral genome is strongly expressed in these cells and the proteins assemble into infectious particles and their co-cultivation with the indicating cells (CEM or M8166) allows the formation of typical syncytia. The HEK293T cells are adherent, cultivated in a monolayer at the surface of the flasks in a MEM medium supplemented with 10% of fetal calf serum (FCS), 1% of penicillin 5,000 Units/ml streptomycin 5,000 μg/ml and 1% of gentamycin 10 mg/ml. The cells are maintained at 37° C. under a humid atmosphere with 5% CO2. The culture medium is changed every three days. In order to carry out sub-cultivations, the nutritional medium is removed, the cells are washed with d PBS/EDTA and incubated for 1 minute at 37° C. in the presence of 0.5% trypsin-0.01% EDTA. After detachment of the cells, a volume of MEM medium is immediately added to the cells and the cells are then homogenized and transferred into new flasks.
The cells CEMx174 and M8166 are human CD4+ T lymphocytes which are permissive to infection by human and simian lentiviruses and form typical cytopathogenic effects (CPE). They are non-adherent and are cultivated in RPMI medium supplemented with 10% of SCS, 1% of penicillin-streptomycin and 1% of gentamycin. These cells are maintained at 37° C. under a humid atmosphere with 5% CO2. The medium is changed every three days by carrying out a centrifugation step at 1,500 G for 5 minutes. The sediment is then re-suspended by successive suctions and discharges into a suitable volume of culture medium.
1.3.2. Functional Evaluation with an In Vitro Biological Test
Transfection of the HEK293T with the Plasmids pCA-LTR-SHIVKU2-IN- or pSHIVKU2 
The transfection method used is the method with ExGen500. ExGen500 (Euromedex, France) consists of a cationic polymer based on linear polyethylenimine. This polymer has a very large cationic charge density allowing it to form complexes with DNA through ionic bonds. These ExGen500/DNA complexes are then capable of interacting with the plasma membranes of the generally anionic cells (interaction via the sulfated proteoglycans). Endocytosis of the complex by the cells ensues as well as their transport towards endosomes/lysosomes. By its protonation capacity at acid pH, ExGen500 gives the possibility of buffering the medium of acid vesicles thereby preventing degradation of the transfected DNA. This property also causes an osmotic shock which allows the DNA to be released in the cytoplasm of the cell. ExGen500 then promotes the transport of the DNA towards the nucleus and avoid its degradation by cytoplasm nucleases.
Five μg of the DNA of the plasmid are added to 350 μl of 150 mM NaCl solution and to 15 μl of ExGen 500. The mixture is incubated for 40 minutes at room temperature. Next, the mixture is added into the flasks containing the HEK-293T covered with freshly renewed medium.
Infection of the CEMx174 and Amplification of the Viral Stock
The HEK-293T transfected with the DNA of pCA-LTR-SHIVKU2-IN- or of pSHIVKU2 are co-cultivated with the CEMx174, and from 48 hours onwards, the CEMx174 develop signs of infection expressed by the formation of ECP which results from the merging of the CEMx174 for forming syncytia. The infected CEMx174 are transferred into a new flask in the presence of fresh CEMx174 for amplifying the virus which is harvested in the supernatant.
Viral Production
The harvesting of the viral stock is carried out from 48 hours onwards by means of a syringe, and the cell debris are then removed by having them pass through a filter with a diameter of 0.22 μm before being put into tubes which are stored at −80° C.
Inoculation of the Cells with the Virus
An aliquot fraction (10-100 μl) of the virus supernatant is used for inoculating the target cells in order to evaluate its infectiousity by cytopathic development or by detection of expression of marker genes.
Titration of the Virus on Non-Adherent Cells
The supernatant containing the virus is diluted in tenfold steps (successive dilutions) in a medium for obtaining dilutions from 10−1 to 10−6 which are used for inoculating in quadruplicate wells containing 1·105 cells per well in 0.5 to 1 ml of RPMI medium in a 24-well plate. The thereby inoculated cells are incubated at 37° C. and with 5% CO2 and sustained by changing medium every 3 days. They are regularly observed for development of ECP.
1.4. Electron Microscopy of HEK293T Cells Transfected with pCA-LTR-SHIVKU2-IN-
With the goal of examining whether the proteins produced by the vaccinal genome pCA-LTR-SHIVKU2-IN- assembled into viral particles, the inventors conducted morphological studies by electron microscopy (EM).
The samples of HEK293T cells transfected with the pCA-LTR-SHIVKU2-IN-, pSHIVKU2 or Δ4SHIVKU2 are fixed in a 2.5% glutaraldehyde solution diluted in a cacodylate buffer (0.1M 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 darkness in uranyl acetate at pH 4. The samples are then immersed in successive baths for 10 minutes of ethanol respectively diluted to 30%, 60%, 90% and 100%. Next, the samples are immersed for two hours in a 50/50 mixture of pure ethanol and of epoxy resin (8 ml of DDSA, 7 ml of MNA, or 13 ml of epoxy). The samples are then placed for two hours in pure epoxy resin before being included in Beem capsules and set to polymerize for 48 hours at 60° C.
Ultrathin cuts of these regions are carried out by means of a diamond cutter with an ultra-microtome. These cuts with a thickness of 70 nm are deposited on copper grids so as to be observed under a voltage of 80 kV by means of a Jeol 1200 EX transmission electron microscope.
1.5. Immunization of Mice with pCA-LTR-SHIVKU2-IN- Vaccinal DNA
1.5.1. Humanization and Vaccination of NOD/SCID Mice
The 6 week old mice are irradiated with a dose of 120 Centigrays of gamma radiation for 50 seconds.
Humanization of the Mice with PBMC of Human Blood
The total sample blood on sodium citrate is centrifuged (2000 g, 10 min, 20° C.) in order to recover the layer of white cells between the plasma and the red cells. The cells are diluted 3 times in PBS/EDTA, delicately deposited above a Ficoll cushion (a medium for separating lymphocytes) and then centrifuged for 45 minutes at 2,000 g at 20° C. The PBMCs are recovered, washed several times in PBS/EDTA and re-suspended in PBSx1 and then 50·106 PBMCs in 0.1 ml are injected for each mouse via an intra-peritoneal route.
Immunization of the Mice
After 48-72 h of post-humanization, the SCID-hu cells are injected via an intra-muscular route (IM) with 50 μg of DNA of the pCA-LTR-SHIVKU2-IN-, pSHIVKU2 or pΔ4SHIVKU2. The BALB/c mice, 6-8 weeks old, are directly immunized by IM injection with 100 μg of each of the DNAs.
1.5.2. Method for Evaluating the Humoral Response
Abnova Sandwich ELISA Test:
This test is based on the detection of the antibodies directed against the viral antigens and which are bound to the bottom of the wells of the 96-well plate. The sera to be tested (recovered at different times post-immunization), the positive and negative controls are deposited in the wells. The anti-HIV Ac (antibodies) optionally present, bind onto the viral antigens. After several washings of the wells for removing the excess and the non-specific bindings, a secondary detection Ac is added which bears a biotin molecule which interacts with a streptavidin coupled to the HPRO enzyme. The formed antigens/Ac complexes will then be detected by adding the substrate of the enzyme, TMB, which will give rise to a colored reaction. The coloration is expressed in optical densities by readout with an ELISA reader photometer.
The reagents and products are brought to room temperature. The negative and positive controls (100 μl) provided in the kit, the blanks (100 μl) and the 10 μl and 50 μl serum samples are deposited in the wells in a final volume of 100 μl. The plate is incubated for 30 minutes at 37° C., and the rinsed with the washing solution. The secondary Ac solution diluted to 1:100 is added (100 μl) in all the wells except in the blanks. The plate is then covered with parafilm paper and incubated for 20 minutes at 37° C. A TMB solution A and B is added after a washing step and the plate is incubated 15 mins at the temperature of the laboratory. For stopping the coloration reaction, 100 μl of 2N H2SO4 are added per well. Readout of the plate is performed at 450 nm.
The samples having an absorbance value equal to or greater than the threshold value are considered to be positive, the threshold value being determined according to the formula: Cutoff Value=NCx+0.100 with NCx=the average of the absorbance values of the two negative controls.
Sero-Neutralization Test
This technique is based on the capability of sero-neutralizing Acs (sero-N) of inhibiting infection of cells sensitive to the virus. For the detection and evaluation of the seric sero-N Acs, a constant amount of infectious viruses is put into contact with serial dilutions of the serum to be tested, and the mixture is then inoculated to a permissive cell culture on microplates and incubated for 3 to 5 days. The virus is most often a cytopathogenic strain, and therefore the absence or the reduction in number of ECPs expresses the presence of Acs in the tested serum.
The viral stock SHIVKU2 is diluted to 1:1000 in RPMI (the supernatant volume of the virus is determined for 100 TCID50, which corresponds to the dilution of a virus for which 50% of the wells have syncytia) and the serums recovered from the NOD/SCID control mice, humanized and vaccinated with pCA-LTR-SHIVKU2-IN- or pSHIVKU2 are diluted in the same medium (at 10, 20, 40, 80, 160 and 320× dilutions). The diluted virus and the serum dilutions are mixed in a 96-well plate (100 μl/well) and the mixture is incubated for 1 h at 4° C. The mixture is then deposited on M8166 cells (1·105 cells/well) cultivated beforehand in a 24-well plate. In this experiment, the positive control is represented by the virus stemming from pSHIVKU2 without any serum and the negative control corresponds to the cells alone.
Method for Evaluating the Cell Response: Elispot Test
This test has the purpose of detecting and evaluating the proportion of T lymphocytes (TL) which secretes IFN-γ as a response specific to antigenic stimulation.
The mice are first deeply anesthetized and then the total blood and the spleen are taken. The spleens of the mice are put in RPMI medium in ice. The blood in dry tubes is used for isolating the serum. The splenocytes are isolated as a result of milling the spleen in a Petri dish between a pair of blades in the presence of PBS and 1% EDTA. The cells are washed twice in PBS/EDTA (centrifugation 2,000G, 5 min at 20° C.) in order to purify and to enrich them with splenocytes. After sowing the wells with the cells, the plate is washed with PBS and then incubated for 30 min with PBS+10% SCS at the temperature of the laboratory. The cells (5·105 splenocytes) are sown in each well, and are inoculated with pools of peptides 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 aluminium foil pouch and incubated at 37° C. for 19 hours. The cells and peptides are washed, and then the biotinylated (7-b6-biotin) anti-IFN-γ monoclonal antibody is added, and the plate is covered and incubated for 2 h at room temperature. The streptavidin diluted in PBS containing 0.5% of FCS is added (100 μl/well) and the plate is incubated for 1 h at room temperature. The TMB, a developer substrate, is added subsequently after another washing step, and the plate is then washed and dried after the emergence of blue spots. Readout of the plate is accomplished with the binocular magnifying glass at a magnification of ×40.
The positivity criteria 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 PBMCs and is normalized to 20% for the data obtained in NOD/SCID mice. The test is considered as being positive if the value of the average of the spots is greater than 10 spots per million of PBMCs which corresponds to the average of spots obtained with the control cultures.
1.6. Phenotype and Functional Examinations of the Specific T Cells of the Antigen by Flow Cytometry
1.6.1. Isolation of the Peripheral Mononuclear Cells
The mononuclear cells of human peripheral blood are prepared as indicated above. The splenocytes of mouse spleens isolated according to the procedure described above are also re-suspended in the AIM V medium without any serum for cultivation and cytometry tests.
1.6.2. Antigenic Stimulation and Cultivation of the Cells
In order to examine whether the specific cells of the antigen are capable of proliferating and of producing cytokines and lytic molecules, the splenocytes and the PBMCs are marked with CFSE (1 μg/ml) for 10 min at 37° C., and the cells are then washed with PBS 1× for removing the excess. The marked cells are sown in deep wells of 96-well plates in an amount of 2·106/well in 1 ml of AIM V medium, and then stimulated with different pools of peptides (Gag, Env and Tat+Rev+Nef) in an amount of 2 μg/ml in the presence of anti-CD49 and CD28 co-stimulation Acs. Cells without any peptides are used as a negative control and cells added with phytohemagglutinin (PHA) at 2 μg/ml are added as a positive control. The cells are cultivated for 5 days (37° C. with humidity) and then re-stimulated with the same pools of peptides for 6 hours before marking them. The cells are harvested by centrifugation (2,000G, 5 min, 4° C.), re-suspended in 100 μl of PBS and first marked with surface Acs (CD3, CD4 and CD8) [Pacific Blue anti-human CD3 (5 μl), PE anti-human CD4 (10 μl) and APC/Cy7 anti-human CD8 (10 μl)] for 30 mins at room temperature. The cells are then centrifuged and washed with PBS 1× and then fixed and made permeable in 100 μl of Cytofix Cytoperm of BD. The cells are then incubated for 20 minutes at 4° C. with anti-human IFN-γ PE-Cy7  and Alexa Fluor 647 anti-human Granzyme A Acs (5 μl) for cytoplasm markings. The cells are finally washed with PBS and fixed with 4% PFA before acquisition and analysis with the flow cytometer.
1.6.3. Instrumentation
An LSRII flow cytometer from BD connected to the BD FACSDiva6 software package was used. This instrument allows measurement of up to 13 fluorescence parameters and two physical parameters which are the FSC size (Forward Scatter) and the complexity or granulosity SSC (Side Scatter). The instrument is equipped with three lasers. The blue laser emitting at 488 nm may independently excite several fluorochromes (FITC, PE, PE-Cy7). The red laser which emits at 633 nm may excite the APC and APC-Cy7 fluorochromes and finally the violet laser which emits at 405 nm may excite the Pacific Blue fluorochrome.
1.7. Immunization of the Macaques
A total of 12 cynomolgus macaques is used in the study. Six macaques form the control group and six other ones form the vaccinated group. The animals were immunized with a single double injection of DNA via the intramuscular route (4 mg/animal) and 1 mg/animal by electroporation (EP).
The reasons for this immunization strategy with a single dose of the vaccine are multiple. One of the main reasons is not to perturb the generation, maturation and amplification of the memory T cells by the primary effector T cells associated with each re-immunization step.
The immunized animals were subject to longitudinal follow up (once a week for 4 weeks and then 1 week out of 2 up to week 32, and then 1 week out of 4 for 10 months) for examining the immune responses induced by the vaccine. The blood samples taken on weeks −2 and −1 before immunization were used for examining possible basal responses. The mononuclear cells of peripheral blood (PBMCs) are isolated and used for evaluating the responses of the T cells with the ELISPOT IFN-λ test, by surface and intracytoplasm markings, and analysis by flow cytometry.
2. Results
2.1. Qualitative and Quantitative Checking of the Construction of the pCA-LTR-SHIVKU2-IN- Plasmid
2.1.1. Presence of the pCA-LTR-SHIVKU2-IN- Plasmid
When the plasmid DNA was isolated from a polyclonal bacterial culture obtained from a preculture in a BL medium used for sowing a large volume of BL liquid medium, a significant proportion of episome is observed around 2000 bp. The electrophoretic profile also shows the presence of a single DNA band around 14,000 bp (which theoretically is 13,739 bp) corresponding to the plasmid.
When the DNA is isolated from a bacterial culture first produced on a Petri dish in order to obtain isolated colonies, these colonies having been used for preculture and bulk culture used for isolating and purifying the plasmid DNA, the electrophoretic profile obtained after separation of 0.5 μg of DNA shows the absence of any episome and shows three bands of high molecular weight DNA corresponding to the circular, wound and superwound forms of the pCA-LTR-SHIVKU2-IN- plasmid. The purity and the quantitative evaluation of the plasmid DNAs of our two preparations were checked by spectrophotometry. The values of the measurements of absorbents at wavelengths 230, 260 and 280 nm were used for determining the 260/280 ratios which were 1.75 and 1.82 and at 260/230 of 2.04 and 1.92 respectively, show satisfactory quality of our DNA. The DNA concentrations are 545 μg/ml and 765 μg/ml, respectively.
2.1.2. Enzymatic Digestion of pCA-LTR-SHIVKU2-IN-
The profile of the digestion of the plasmid with EcoR1 reveals the presence of two bands of about 5,000 and 7,500 bp from cuts at both EcoRI sites, three bands with Bam H1 of 2400 bp, 4,900 bp and 7,400 bp resulting from cuts at both Bam H1 sites, and a band with Sph1 located at 10,000 bp resulting from the cut at the single Sph1 site. An additional band of 2,400 bp is also observed with the BamH1 digestion and it would result from the episome.
2.2. Evaluation of the Functionality: Effect of the Plasmid DNA on the Cells
The HEK293T cells were transfected with a control plasmid pCG-GFP expressing GFP, the plasmid pSHIVKU2, and then with the plasmid pCA-LTR-SHIVKU2-IN-. The cells transfected with the plasmid pCG-GFP gave the possibility of estimating the efficiency of transfection by evaluating the number of GFP+ cells.
In order to check that the HEK293T cells transfected with pCA-LTR-SHIVKU2-IN- or pSHIVKU2 produce virions which induce typical syncytia, these cells were co-cultivated with CEMx174, and the occurrence of the ECPs was then followed by observation under the microscope. Both co-cultures produced characteristic ECPs.
In order to check whether the SHIVKU2 virus produced by the transfected cells replicates several times, the supernatant of the transfected cells was used for infecting the M8166 human CD4+ T lymphocyte line. The results obtained with the SHIVKU2, clearly show that it infects and induces ECPs especially with the highly permissive M8166 cell line. These ECPs appear as soon as after 48 hours but also at belated stages.
Next, in order to check that the DNA of the vaccinal vector pCA-LTR-SHIVKU2-IN- only allows a single replication cycle (as it is deleted from the in gene), the recovered supernatant containing the CA-LTR-SHIVKU2-IN- virus was inoculated a first time, and then a second time to M8166 cells being cultivated. The first inoculation produced ECPs from 48 hours onwards and at the end of 76 hours they were more numerous. The supernatant of these infected cells was recovered and inoculated once again to M8166. Unlike the previous inoculation, no ECP is visible either 48 or at 76 hours after inoculation. These results allow the conclusion to be drawn that pCA-LTR-SHIVKU2-IN- gives the possibility of transducting only once the target cells being cultivated.
The presence of typical ECPs, suggest that the plasmid DNA is replicated in the HEK293T cells and has produced CA-LTR-SHIVKU2-IN- virions. The ECPs of the first infection show the infectivity of the M8166 human CD4+ TL line by the produced particles, and their absence during the second infection shows the productive replication deficit in the cells.
2.3. Electron Microscopy Analysis of the Morphogenesis of Viral Particles in HEK293T Cells Transfected with pCA-LTR-SHIVKU2-IN-
It was next determined whether the proteins produced by the vaccinal pCA-LTR-SHIVKU2-IN- genome properly assembled into viral particles in the HEK293T cells. The morphology of the HEK293T cells transfected with the plasmid pCA-LTR-SHIVKU2-IN- was examined by EM.
The results showed that the viral particles are present at the surface of the cells in the form of buds and mature viral particles which have detached from the cells. Thus, the viral proteins of the pCA-LTR-SHIVKU2-IN- vaccine assemble in order to give viral particles which bud on the outside of the cells.
2.4. In Vivo Test of the Vaccine on Humanized SCID Mice and on BALB/c Mice
2.4.1. Evaluation of the Humoral Response in SCID-Hu Mice Vaccinated with pCA-LTR-SHIVKU2-IN-
The serum samples of the immunized mice, taken at about 1 month after immunization are examined for the presence of Acs which specifically bind to the antigens of the virus by means of a commercial ELISA test. The results of this analysis are summarized in Table 1 (below). These results show that about half of the samples from the mice immunized with vaccinal pCA-LTR-SHIVKU2-IN- DNA, like those immunized with DNA of SHIVKU2, have lesser proportions of positives (44% and 37%, respectively) unlike those from the mice immunized with pΔ4SHIVKU2 (50%). These results demonstrate the capability of the vaccinal pCA-LTR-SHIVKU2-IN- DNA of inducing humoral responses in NOD/SCID-hu mice. One sample out of three of serum of the immunized mice with pΔ4SHIVKU2, has its OD value greater than those obtained with the samples of serums of immunized mice with pCA-LTR-SHIVKU2-IN- and pSHIVKU2. This plasmid is non replicative, the proteins of the virus remain associated with the membrane of the transfected cells and therefore should induce less Acs.







TABLE 1







Detection of antibodies in samples of sera of control SCID, SCID-hu and SCID-


hu mice, immunized with pΔ4SHIVKU2, pCA-LTR-SHIVKU2-IN- and pSHIVKU2 and


of the controls.









Samples
















CA-LTR









SHIVKU2-


Positive
Negative




Δ4SHIV
IN-
SHIV
controls
controls
controls
Blanks





Number of
6
16
19
2
2
2
2


samples









Positivity
3
 7
 7
2
2
2
2


number









Absorbance
0.3-1.823
0.26-0.9
0.34-1.64
0.01-0.19
1.9-2.2
0.15-0.16
0.01-0.03


positivity









Interval









2.4.2. Analysis of the Neutralizing Activity by Sero-Neutralization

The serums of SCID-hu mice vaccinated with the selected plasmids pCA-LTR-SHIVKU2-IN- or SHIVKU2 are those for which the OD was found positive by ELISA. Because of the small amount of serum, certain samples with a strong OD value for the ELISA test, were not able to be examined by sero-neutralization. A serum sample of SCID-hu mice and vaccinated with pCA-LTR-SHIVKU2-IN- found to be negative was also used for the ELISA test for ensuring the reliability of the test.







TABLE 2







Analysis of the neutralizing activity of the sera of immunized mice.


Dilutions ( 1/10, 1/20, . . . 1/320) were mixed with the virus


SHIVKU2 (100 TCID50), incubated and then used for inoculating the


M8166 cells. After 5 days after inoculation, the induced EPCs are


listed and used for evaluating the sero-neutralizing activity.


Caption: +++ correspond to strong neutralization, ++ quite high


neutralization, + less neutralization, − no neutralization action.


For the CA-LTR-SHIVKU2 etc, two types of samples are represented, a


type of sample having a less neutralizing profile as compared with


the two other samples.


Samples








CA-LTR-SHIVKU2-IN- (3 samples)
SHIV (1 sample)











Dilutions
Neutralization
ECP interval
Neutralization
ECP interval














10
+++/+++
5-9
+++
4


20
++/++
14-19
++
12


40
+/−
25-44
++
20


80
+/−
27-49
++
15


160
+/−
31-57
+
26


320
+/−
28-69
−
44









The number of ECPs obtained is higher in the M8166 cells infected by the SHIVKU2 virus incubated without any serum (76 ECPs) or with the serum of the non-immunized mice (42 ECPs) (negative control), which allowed us to set the negativity threshold of viral neutralization to 42 ECPs (data not shown in the table). On the other hand, the number of ECPs becomes quasi zero when the virus is incubated with 1/10 diluted serum of mice immunized with the DNA of CA-LTR-SHIVKU2-IN- or SHIVKU2. This number increases gradually as increases the dilution of the serums indicating a dose effect. For example, with the serum of a mouse immunized with pCA-LTR-SHIVKU2-IN- at 1/10 dilution, 5 ECPs are obtained, whereas at 1/320 dilution, a value of 69 ECPs is obtained, a value similar to the one of the control without any serum.
2.4.3. Evaluation of the Immunogenicity of pCA-LTR-SHIVKU2-IN- in Vaccinated BALB/c Mice
In order to study the immunogenicity of the pCA-LTR-SHIVKU2-IN- vaccine in BALB/c mice, the animals were injected with a single dose of 100 μg of DNA via an intramuscular route. The proportion of specific spleen cells (splenocytes) of the antigens was examined by ELISPOT. The results of this study actually show the capability of the DNAs used of inducing specific immune responses directed against all the studied antigens (FIG. 12). The T cell responses with the plasmid pΔ4SHIVKU2 are twice higher than with the plasmid pCA-LTR-SHIVKU2-IN-. This small difference in efficiency between both DNAs may be related to better quality of the DNA of pΔ4SHIVKU2 than that of the pCA-LTRSHIVKU2-IN-. These results nevertheless give the possibility of determining the base level of the immune responses induced by these vaccines in normal mice and give the possibility of performing a comparison with the immune responses obtained in the SCID-hu mouse.
2.4.4. Evaluation of the Cell 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 μg of DNA of CA-LTR-SHIVKU2-IN-, Δ4SHIVKU2 or SHIVKU2, and the splenocytes were then used for examining the immune response by ELISPOT for evaluating the proportion of specific cells of the antigen and producing IFN-γ. As shown in FIG. 13, there is a significant number of T cells producing human INF-γ in response to stimulation by the 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-SHIVKU2-IN- are quasi-similar to those obtained with pSHIVKU2 and which are both clearly greater than those obtained after immunization with pΔ4SHIVKU2. The predominancy of the responses induced by pCA-LTR-SHIVKU2 is against the Gag and Tat+Rev+Nef antigens.
The whole of these results demonstrates that the DNA of pCA-LTR-SHIVKU2 is highly immunogenic in NOD/SCID-hu mice and that it preferentially induces responses against the antigens known for being associated with the protection against pathogenic viruses.
2.5. Phenotype and Functional Examinations of Specific T Cells of the Antigen by Flow Cytometry
2.5.1. Monomarking Carried Out on Day Zero (Performed on the Day when the Mouse Spleens were Recovered)
These mono-markings are used in order to examine whether the selected antibodies actually detect those targets on the one hand, and for evaluating the presence and the proportion of human cells in spleens of SCID-hu mice on the other hand.
The non-marked lymphocytes of humanized and non-humanized mice were analyzed in flow cytometry for measuring the fluorescence in the basal state of the cells (negative control).
The detection of CD3+ TLs is carried out with human anti-CD3 Acs coupled with the fluorochrome “Pacific Blue”. These cells form a peak at 102 in the profile of cells isolated in SCID-hu cells vaccinated with the plasmid pCA-LTR-SHIVKU2-IN-. This peak is not present in the cells recovered in SCID-non-hu mice.
No significant peak was observed for cells mono-marked with anti-CD4+ Ac (CD4 anti-human PE), whether this is for the control or for our tested sample. This would be due to the anti-CD4 Ac used which would be non-functional.
For detecting CD8+ TLs, a human anti-CD8 monoclonal Ac coupled with the fluorochrome APC/Cy7 was used. It allows detection of a peak located between 102 and 103 in the profile of the cells isolated in SCID-hu mice vaccinated with pCA-LTR-SHIVKU2-IN- and not in SCID-non-hu mice.
The proportion of lymphocytes producing GRA molecules was not able to be evaluated because no peak difference was observed on the cells from immunized and non-immunized mice marked with anti-GRA Ac coupled with Alexa Fluor 647.
On the other hand, the cells marked with human anti-IFN-γ Ac coupled with the fluorochrome PE-Cy7 showed a clear peak located at 102 with cells of SCID-hu mice vaccinated with pCA-LTR-SHIVKU2-IN- which is absent with cells of non-immunized SCID-non-hu mice.
2.5.2. Phenotyping and Functions of the T Cells in Immunized Animals
In order to examine the phenotype and the functions of the specific T cells of the antigens of the virus, the cells marked with CFSE are incubated for five days with the (Gag, Env and Tat+Rev+Nef) peptides and then re-stimulated for 6 hours with the same peptides. The cells are then marked with the Acs and examined as indicated 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 the cells from vaccinated NOD/SCID-hu mice. Indeed, at least 6% of the CD8+ TLs produce IFN-γ and 1.7% produce GRA A in immunized NOD/SCID-hu mice, versus only 2.5% and 0.6% respectively in control mice. These results demonstrate the presence of CD3+, CD8+ activated cells which produce GRA and IFN-γ, which corresponds to an effector cellular immune response induced by our vaccine pCA-LTR-SHIVKU2-IN-.
2.6. Analysis of the Immune and Humoral Responses in Macaques
2.6.1. Analysis of the Immune Responses of T Cells by the ELISPOT IFN-λ Test
A fraction of the mononuclear cells isolated from blood samples taken was used for evaluating the proportion of cells secreting IFN-λ in response to stimulation by the viral Gag, Pol, Env and Tat+Rev+Nef antigens by means of a commercial kit. The results of the first 20 weeks of the analysis are shown in table 3. They clearly show that following a single administration of the vaccinal vector CAL-SHIV-IN-, all the animals developed cellular immune responses characterized by specific cells of the antigens, which secrete IFN-λ. These responses are heterogenous according to the animals which have CMH-1 haplotypes different from each other. The immune responses are characterized by the presence of a first primary response peak at 2-4 weeks post-immunization (PI), and then of more belated responses from 8-10 weeks PI, and this in the absence of a second immunization. It is of great interest to note that the intensity of the second peak is often much greater than that of the first peak especially for the BX80 animal where the number of cells secreting IFN-λ is multiplied by 3.







TABLE 3







Summary of the proportions of T cells secreting gamma interferon cytokine (IFN-λ)


in response to the stimulation by the viral antigens


(Gag, Pol, Env and Tat + Rev + Nef) expressed by the vaccine in vaccinated


monkeys. The figures correspond to the numbers of secreting cells forming one 


spot per million (106) of mononuclear cells of peripheral blood (PBMC).


The weeks of analysis are indicated at the top of the table.









Post-Immunization week




















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









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























BX80
Gag
208
821
666
472
317
504
538
474
226
574
621
862



Pol
0
105
17
350
15
32
0
97
27
11
11
168



Env
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


BX83
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



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



TRN
0
51
204
116
0
0
731
393
195
224
143
432


BX84
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



Env
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


BX72
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



Env
7
79
57
56
12
168
56
5
0
0
12
132



TRN
5
35
161
192
92
692
445
736
0
25
119
587


BX78
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



Env
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









2.6.2. Analysis of the Immune Responses of T Cells by Multiparameter Flow Cytometry in Vaccinated Monkeys

The results of the analysis by multiparameter flow cytometry will confirm those obtained by ELISPOT by revealing that all the animals developed a response consisting of T cells which proliferate and which are specific of all the antigens expressed by the vaccinal vector (Table 4). These responses are heterogenous among the animals and also reveal a first primary response phase which extends up to about 8 weeks PI, followed by a contraction phase (2-4 weeks) and then by a re-emergence phase. This longitudinal tracking of the immune response by multiparameter flow cytometry is continued until the virulent test with the test virus SIVmac251 which is conducted in week 52.







TABLE 4







Summary of the values of the CD4+ and CD8+ proliferating


T cells in response to the antigenic stimulations at the moment of


the primary expansion phases, of the contraction and finally re-


emergence or secondary expansion phases in vaccinated


monkeys. The weeks corresponding to each of the phases


are indicated. The numbers correspond to the percentages


of specific T cells of each of the antigens which proliferate


in response to the stimulation as compared with the total


number of T cells.











Proportion of T cells which proliferate in


Animals
Ag
response to the antigens (Ag)



















Week
Week
Week
Week




Type
(−1)
(3-6)
(8-20)
(22-26)





BX80
Gag
CD8+
0.5
3.6
0.4
1.5




CD4+
0.1
0.1
0.1
0.4



Pol
CD8+
ND
0.6
0.2
0.7




CD4+
ND
0.7
0.1
0.6



Env
CD8+
0.3
0.5
0.1
1.4




CD4+
0.5
1.3
0.2
0.5



TRN
CD8+
0  
4.8
0.8
1.6




CD4+
0.3
1.2
0.2
0.5








Week
Week
Week
Week




Type
(−1)
(2)
(8-20)
(22-26)





BX83
Gag
CD8+
0.2
1.7
0.5
1.6




CD4+
0.1
0.6
0.4
0.9



Pol
CD8+
ND
ND
0.3
0.7




CD4+
ND
0.3
0.2
0.5



Env
CD8+
0.1
ND
0.5
1.2




CD4+
0.3
ND
0.3
0.7



TRN
CD8+
0.4
1.5
0.6
2.3




CD4+
0.2
0.8
0.3
0.5








Week
Week
Week
Week




Type
(−1)
(3-6)
(8-22)
(24-26)





BX84
Gag
CD8+
0.1
1.1
0.3
2.6




CD4+
0.1
0.3
0.1
0.8



Pol
CD8+
ND
0.3
0.2
0.7




CD4+
ND
0.3
0.2
0.5



Env
CD8+
0.1
1.1
0.3
0.9




CD4+
0.2
0.8
0.3
0.7



TRN
CD8+
0  
1.3
0.7
1.1




CD4+
0.2
0.8
0.3
0.5








Week
Week
Week
Week




Type
(−1)
(3-6)
(8-20)
(22-26)





BX72
Gag
CD8+
0  
4.2
0.7
3.0




CD4+
0  
0.6
0.2
0.4



Pol
CD8+
ND
0.7
0.2
0.7




CD4+
ND
1.3
0.1
0.1



Env
CD8+
0.2
1.0
0.5
0.6




CD4+
0.3
1.4
0.5
2.3



TRN
CD8+
0.1
0.9
0.3
1.5




CD4+
0.1
1.7
0.4
0.9








Week
Week
Week
Week




Type
(−1)
(3-6)
(8-18)
(20-32)





BX78
Gag
CD8+
0  
3.4
1.1
3.7




CD4+
0.1
1.3
0.6
2.9



Pol
CD8+
ND
1.1
0.3
1.1




CD4+
ND
0.6
0.2
1.3



Env
CD8+
0.2
1.4
0.4
1.2




CD4+
0.3
0.6
0.6
2.5



TRN
CD8+
0.3
1.9
0.8
2.1




CD4+
0.3
1.2
0.5
2.1









2.6.3. Analysis of the Humoral Response in Macaques

Detection of the anti-antigen antibodies of SHIV was carried out with a commercial ELISA kit which allows detection of the anti-Env antibodies of the HIV-1. The longitudinal examination of the serums harvested at each blood sampling point showed the presence of anti-Env antibodies from week 20 PI in the BX80 animal and from week 8 for the BX73 animal. The presence of antibodies in positive sera was confirmed by Western blot against proteins of the SHIV showing a strong signal against the Gag-p27 protein as well as a signal against the gp160/gp120 glycoproteins.
2.6.4. Conclusion
These results clearly demonstrate that a single injection of vaccinal DNA (CAL-SHIV-IN-) gives the possibility of inducing T cell and humoral (antibody) immune responses. The T cell responses are directed against all the viral antigens expressed by the vaccinal vector. They are persistent and follow a conventional expansion, contraction and storage-in-memory scheme. The presence of memory T cells of the central type and of memory effector cells was confirmed by the phenotyping.
2.7. Construction and Functional Analysis of a Novel Vector Expressing the Whole of the Antigens of the HIV-1: The Vector CAL-HIV-IN-
From the genome of the vector CAL-SHIVKU2-IN-, the gag, pol, vif, vpx and vpr genes of the SIV were deleted following double digestion with the Nar1 and Sph1 enzymes, and the nef gene of the SIV was deleted following the partial digestion with the Nru1 enzyme and the total digestion with the Not 1 enzyme. The remaining fragment was then purified. This fragment, bearing the tat, rev and env genes of the HIV framed with the RTLs of the CAEV and transported by the plasmid pET, was used for introducing the 5 kb fragment bearing the gag, pol, vif, vpx and vpr genes of the HIV-1 and the 620 bp fragment bearing the nef gene of the HIV-1, and generating the vector CAL-HIV-IN- (FIG. 17). The CAL-HIV-IN- vector, deleted from the sequences coding for the integrase, consists in the sequence SEQ ID NO: 18.
2.7.1. Evaluation of the Functionality: Effect of Plasmid DNA on the Cells
The DNA of this vector was introduced into the GHOST-CXCR4 and HEK-293 T cells by transfection using the ExGen and the procedure recommended by the manufacturer. The transfected GHOST-CXR4 cells had then become fluorescent confirming expression of the viral proteins of the HIV-1 by the vaccinal vector and more particularly the Tat protein which transactivates the expression of the GFP (Green Fluorescent Protein) gene under the control of the RTL of the HIV.
The supernatant of the HEK-293T cells transfected with the vaccinal vector CAL-HIV-IN- was used for inoculating the M8166, indicating CD4+ T cells which developed cytopathic effects, characteristics of HIV infection. These results provide the proof that the proteins expressed by the vaccinal vector assembled into viral particles allowing infection of the M8166 indicator cells. These cells with the cytopathic effects did not produce any viruses capable of again infecting the M8166 indicator cells and inducing cytopathic effects. This result indicates that CAL-HIV-IN- is associated with a single replication cycle in the absence of integration.
In order to evaluate the viral proteins produced after transfection, the supernatants of the transfected HEK-293T cells with the vaccinel vector, were harvested 24 h, 48 h and 72 h post-transfection and then examinated for the presence of Gag p24 antigens with ELISA. The measurements of the amounts of this protein are indicated in Table 5. They show increasing accumulation of this protein ranging from 100 ng/ml at 24 h up to 135 ng/ml at 72 h post-transfection.







TABLE 5







Evaluation of the Gag p24 protein of the HIV-1 secreted in the


supernatant of HEK 293T cells transfected with the vaccinal


vector CAL-HIV-IN-. Quantification of the protein p24


accumulated in the supernatant of the HEK 293T cells after


24, 48 and 72 hours post-transfection with the DNA of the


vector CAL-HIV-IN-.











24 h
48 h
72 h















Gag p24 concentration
100 ng/ml
110 ng/ml
135 ng/ml









2.7.2. Immunization of BALB/C Mice and Characterization of the Induced Immune Responses

Three groups of BALB/c mice (6 per group) of 6 weeks old were used: two groups were used for immunization and the third group is a control group. The two groups of immunized mice were injected with 100 μg/mouse DNA of the vector CAL-HIV-IN- via the intramuscular route. The animals of a group were sacrificed after two weeks and the other ones at three weeks post-immunization (PI). The control mice were sacrificed at two weeks PI. The spleens of each of the mice were taken, the splenocytes were isolated and then used either for the ELISPOT test or for the analysis in multiparameter flow cytometry as described above. The results of the analysis by ELISPOT are indicated in Table 6. They show the presence of specific cells of all the antigens which secrete IFN-λ. The majority of these cells are specific of Gag and Tat+Rev+Nef antigens. The responses at 2 and 3 weeks post-immunization are substantially similar.







TABLE 6







Summary of the results of the analysis by ELISPOT on the


splenocytes of BABL/c mice immunized with the vaccinal


vector CAL-HIV-IN- at 2 and 3 weeks post-immunization.


The isolated splenocytes of the mouse spleens immunized


with 100 μg per mouse via an intramuscular route were


examined with the ELISPOT IFN-λ test for evaluating


the number of T cells secreting the cytokine in response to


the stimulations with the pools of peptides (Gag, Env and


Tat + Rev + Nef, TRN). The averages of the number


of spots for each antigen and for the 6 examined mice after


2 and 3 weeks post-immunization are indicated.












Medium without any






peptide
Gag
Tat + Rev + Nef
Env














2 weeks
10
45
50
17


3 weeks
5
35
55
15









The results of the analysis by flow cytometry (Table 7) demonstrate specific CD4+ and CD8+ T cell immune responses of all the antigens expressed by the vaccinal vector CAL-HIV-IN-.







TABLE 7







Summary of the results of the analysis by multiparameter flow


cytometry. The figures correspond to the percentages of


specific T cells of each of the antigens which proliferate in


response to the antigenic stimulation, based on the total


number of T cells.












Medium without






any peptide
Gag
Tat + Rev + Nef
Env














CD3 + CD4+
0.16
0.26
0.40
0.18


CD3 + CD8+
0.20
0.27
0.50
0.25

## Claims

1. A nucleic acid comprising a chimeric retroviral genome, in which said chimeric retroviral genome comprises:
Long terminal repeat sequences (LTR) positioned 5′ and 3′ of a first retrovirus, said first retrovirus being the Caprine Arthritis Encephalitis Virus (CAEV), and
at least one viral gene of a second retrovirus, said second retrovirus not being the first retrovirus,
wherein the chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said second retrovirus, and
wherein said pol gene is mutated to delete nucleic acid sequences encoding an integrase protein to an extent sufficient to abolish any integrating activity of said integrase protein.

2. The nucleic acid according to claim 1, wherein said second retrovirus is a lentivirus.

3. The nucleic acid according to claim 2, wherein said lentivirus is selected from the human immunodeficiency virus of type I (HIV-1), the HIV-2, the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV), or the equine infectious anaemia virus (EIAV).

4. A recombinant vector comprising the nucleic acid according to claim 1.

5. An immunogenic or vaccinal composition comprising the nucleic acid according to claim 1.

6. A nucleic acid comprising a chimeric retroviral genome, in which said chimeric retroviral genome comprises:
Long terminal repeat sequences (LTR) positioned 5′ and 3′ of a first retrovirus, said first retrovirus being the Caprine Arthritis Encephalitis Virus (CAEV), and
at least one viral gene of a second retrovirus, said second retrovirus not being the first retrovirus;
wherein 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 retroviruses; and
wherein the chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu, and env genes each of said genes being of either but not both of said second or third retrovirus, and
wherein if said at least one viral gene of said second retrovirus or said at least one viral gene of said third retrovirus is a pol gene then said pol gene is mutated to delete nucleic acid sequences encoding an integrase protein to an extent sufficient to abolish any integrating activity of said integrase protein.

7. The nucleic acid according to claim 6, wherein said at least one gene of said second retrovirus, or said at least one gene of said third retrovirus, is selected from the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes.

8. The nucleic acid according to claim 6, wherein the chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef genes of said second retrovirus and the tat, rev, vpu and env genes of said third retrovirus.

9. The nucleic acid according to claim 6, wherein said second and third retroviruses are each a lentivirus.

10. The nucleic acid according to claim 9, wherein said lentivirus is selected from the group consisting of human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), or equine infectious anaemia virus (EIAV).

11. The nucleic acid according to claim 6, wherein said retroviral chimeric genome comprises the gag, pol, vif, vpx, and vpr genes of SIV and the nef, tat, rev, vpu and env genes of HIV-1.

12. The nucleic acid according to claim 6, wherein said retroviral chimeric genome comprises at least one of the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of HIV-1 and comprises at least one of the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of HIV-2.

13. The nucleic acid according to claim 6, wherein said second retrovirus is a lentivirus.

14. The nucleic acid according to claim 6, wherein said chimeric retroviral genome comprises one of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6, and
wherein said pol gene, when it is present, is mutated to delete nucleic acid sequences encoding an integrase protein to an extent sufficient to abolish any integrating activity of said integrase protein.

15. A recombinant vector comprising the nucleic acid according to claim 6.

16. An immunogenic or vaccinal composition comprising the nucleic acid according to claim 6.

17. A nucleic acid comprising a chimeric retroviral genome, wherein said chimeric retroviral genome comprises one of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6.