CN103732248A - Pharmaceutical compositions for preventing and/or treating an hiv disease in humans - Google Patents
Pharmaceutical compositions for preventing and/or treating an hiv disease in humans Download PDFInfo
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- CN103732248A CN103732248A CN201280026590.4A CN201280026590A CN103732248A CN 103732248 A CN103732248 A CN 103732248A CN 201280026590 A CN201280026590 A CN 201280026590A CN 103732248 A CN103732248 A CN 103732248A
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-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention relates to pharmaceutical compositions comprising a mixture of a specific HIV antigen and a non-pathogenic living bacterium. Said specific HIV antigen comprises one or more epitopes from Gag and/or Pol proteins and is preferably under a particulate form. Said bacterium is preferably Lactobacillus plantarum. These compositions are useful for preventing and/or treating an HIV disease in humans.
Description
Technical field
The pharmaceutical composition of the mixture that the present invention relates to contain specificity HIV antigen and non-pathogenic bacteria.Described specificity HIV antigen comprises one or more epi-positions from Gag and/or Pol albumen, and preferably its under the form of granule (particulate).Described antibacterial is Lactobacillus plantarum (Lactobacillus plantarum) preferably.These compositionss are useful to the HIV disease preventing and/or treating in people.
Background technology
Rear over 25 years at finder's immunodeficiency virus (HIV), up-to-date prediction (projection) from the United Nations's co-plan of World Health Organization (WHO) and HIV/AIDS shows, if be very popular with current speed progress, by 2011, will had and surpass 3,000 ten thousand people's infection.
But, although in order to find that pre-preventing HIV infection is effectively treated and made a large amount of research effort, the preventative vaccine of order first two test or failure people such as (, 2008) Mc Elrath or produce moderate effect people such as (, 2009) Rerks-Ngarm.
People (the Clinical and Vaccine Immunology such as Jae-Sung Yu, 2006 November, 13 volumes, No.11,1204-1211) described restructuring smegma mycobacteria (Mycobacterium smegmatis) carrier, it builds, and for expressing, conduct is surperficial, cell is interior or the total env gene C ON6 of M group HIV-1 of secretory protein.Author can prove in mice, and restructuring smegma mycobacteria (M.smegmatis) is to being immunogenic in mucomembranous surface induction HIV-1T cell effect.
The people such as Ke-Qin Xin (Blood, 2003 June 1,102 volume, No.1,223-228) Recombinant Lactococcus lactis (Lactococcus lactis) carrier at its cell surface expression HIV-1Env V2-V4 ring described.With this carrier oral immunity mice, induce:
-mucosa and humoral immune reaction, as by detecting as indicated in high-caliber HIV specific serum IgG and Excreta IgA antibody; With
-cell immune response, as indicated in the quantity increase by HIV specificity IFN γ secretory cell.
For at lactococcus lactis (L.lactis) cell surface correction, can use 1kb or be less than the genetic fragment (segment) of 1kb.
Most of scientist's sensations that participate in HIV pathogeny and prevention are before test is used for preventing or treats the HIV preventative vaccine or other biological composition of mankind HIV infection, in inhuman Primates, test its homologue by more constructive meaning (Morgan C, Deng people, 2008).The inhuman Primates of selecting are Rhesus Macacus (macaques rhesus); in macaque (macaques); the macaque that has shown for certain now the China source of infected monkey immunodeficiency virus (SIV) 239 is that in simulation majority, HIV infects the best model of develop clinical, virusology and immunology (people such as Marcondes MC, 2006; The people such as Stahl-Hennig C, 2007; The people such as Chen S, 2008).
Finally, scientific circles' approval now, once find the effective preventative biological composition of anti-SIV239 or vaccine in macaque, it should may successfully be applicable to people completely and protect it to avoid AIDS.
Although the research effort that scientific circles are lasting, still expects that the preventative and effective strategy of therapeutic defeats global AIDS popular.
Describe various antibacterials and there is interesting adjuvanticity while being applied to experimenter and the characteristic of immune regulative.Especially, reported that lactic acid bacteria promotes immune tolerance effect.
For example, be published on November 23rd, 2006, at Stallergenes S.A WO2006/123230 under one's name, described and be selected from the antibacterial of bacillus bifidus (Bifidobacteria) and lactic acid bacteria as the purposes of the adjuvant in immunogenic composition, inducing antigen-specific tolerance when it can be in Sublingual, through tongue or by oral administration to experimenter.Suggestion immunogenic composition is used for the treatment of allergy, autoimmune disease or for preventing transplant rejection.
Yet, for example be published on July 30th, 2009, at food and nutrition Advanced Study Institutes (Stichting Top Institute Food and Nutrition) WO2009/093900 under one's name, the tolerogenesis compositions that contains a large amount of mid-log phase lactic acid bacterias has been described.When being applied to experimenter, said composition is induced non-antigen specific immune tolerance.Suggestion said composition is used for preventing, delay and/or treat disease or the disease relevant to the inflammatory reaction that causes tissue injury, for example, and the inflammation disease of allergy, autoimmune disease and intestinal.
Summary of the invention
Astoundingly, inventor can show that original pharmaceutical composition as described in following examples induces the effectively antigen specific immune protection to SIV in macaque.And when the protection of described SIV specific immunity is induced, inventor shows that it has prevented SIV to copy/propagate and the foundation of In vivo infection subsequently.
In fact, when inventor shows in surprise disclosed pharmaceutical composition mucosa ground in literary composition or used by Intradermal or upper intradermal routes, suppress significantly or even eliminate or prevent virus replication.
In fact, inventor observes the early stage activation of the CD4+T cell of non-cytotoxicity CD8+T cell effect inhibition SIV angtigen presentation in macaque for the first time.Thereby, be not wishing to be bound by theory, when mucosa or Intradermal or upper Intradermal are applied to experimenter, pharmaceutical composition according to the present invention is induced beyond thought new virus specific immunologic tolerance.This immunologic tolerance look like HIV Gag and/or Pol antigenic specificity inhibition CD8+T cell induction immunologic tolerance (in literary composition also called after "
t presses downproperty processed " " Ts " immunologic tolerance of immunologic tolerance), it is that MHC(is " ajor histocompatibility complex ")-1b/E restriction with non-cytotoxicity.
In view of the result of reporting in literary composition, the invention provides a kind of new pharmaceutical composition that can realize as defined above " Ts " immunologic tolerance, it is for preventing and/or treating people's HIV disease.
Thereby, an object of the present invention is to provide the pharmaceutical composition of the mixture that contains antigen and non-pathogenic antibacterial alive, wherein, preferred described antigen be granule and/or its there are one or more epi-positions from HIV Gag and/or Pol albumen, and wherein said antibacterial Lactobacillus plantarum preferably.
Another object of the present invention is to provide the pharmaceutical composition described in literary composition, as vaccine.
Another object of the present invention is to provide for preventing and/or treating the method for the people's who needs HIV disease, and it comprises at least mucosa, and (preferred oral) or Intradermal ground or upper Intradermal ground are applied to the aforementioned pharmaceutical compositions of effective dose described people's step.
Another object of the present invention is to provide the method for avoiding HIV for the protection of people, and it comprises mucosa at least, and (preferred oral) or Intradermal ground or upper Intradermal ground are applied to the aforementioned pharmaceutical compositions of effective dose described people's step.
Another object of the present invention is to provide the method for avoiding HIV seroconversion for the protection of people, and it comprises mucosa at least, and (preferred oral) or Intradermal ground or upper Intradermal ground are applied to the aforementioned pharmaceutical compositions of effective dose described people's step.
Another object of the present invention is to provide for preventing and/or treating the pharmaceutical kit of the people's who needs HIV disease, and it comprises
-antigen in the first container; With
-non-pathogenic bacteria in second container,
Wherein said antigen and described antibacterial for mucosa or Intradermal or on the pharmaceutically acceptable carrier of intradermal administration, wherein preferred described antigen be granule and/or its there are one or more epi-positions from HIV Gag and/or Pol albumen, and wherein said antibacterial preferred plant lactobacillus.
Accompanying drawing explanation
By following accompanying drawing explanation the present invention, it can be with reference to following unrestriced embodiment.
Fig. 1: with intravenous (i.v.) the SIVmac239 challenge of the pretreated Rhesus Macacus of intravaginal iSIV/BCG.
Fig. 2: with internal rectum (i.r.) the SIVmac239 challenge of the pretreated Rhesus Macacus of intravaginal iSIV/BCG.
Fig. 3: with the repetition SIVmac239 challenge of intravaginal iSIV/BCG pretreated Rhesus Macacus (3 times by i.v. and 2 times by i.r.).
Fig. 4: add the intravenous SIVmac239 challenge that Intradermal is strengthened pretreated Rhesus Macacus with intravaginal iSIV/BCG.
Fig. 5: add the internal rectum SIVmac239 challenge that Intradermal is strengthened pretreated Rhesus Macacus with intravaginal iSIV/BCG.
Fig. 6: with the internal rectum SIVmac239 challenge of the pretreated Rhesus Macacus of oral iSIV/BCG.
Fig. 7: from the CD8+T cells in vitro antiviral activity obtaining with the pretreated Rhesus Macacus of intravaginal iSIV/BCG.
Fig. 8: from the CD8+T cells in vitro antiviral activity obtaining with pretreated 4 Rhesus Macacus of oral iSIV/BCG.
Fig. 9: the SIV specificity of the CD4+T cell-stimulating by autologous homology (autologous) the CD8+T cell from obtaining with pretreated 4 Rhesus Macacus of oral iSIV/BCG suppresses.
Figure 10 a: take from the anti-SIV IgG antibody titer in the plasma sample of iSIV/LP, iSIV or the pretreated Rhesus Macacus of LP.
Figure 10 b: take from by the SIV specific T-cells propagation in the PBMC sample of iSIV/LP, iSIV or the pretreated Rhesus Macacus of LP.
Figure 10 c: SIV specificity IFN γ secretion T cell under stimulating in vitro when existing or not having CD8 or CD25T cell.
Figure 10 d: with use oral LP(n=4) or iSIV(n=3) pretreated animal compare, the SIV specificity of the CD4+T cell-stimulating by the autologous homology CD8+T cell that obtains with pretreated 8 Rhesus Macacus of oral iSIV/LP suppresses.
Figure 10 e: gastric is used after iSIV/LP preparation the SIV specific C D8+T cell of 60 days: at CD8+T cell or while existing the K562 of people's natural killer cell (hNK) (contrast) to exist, when thering is or not having SEB and anti-CD3/CD28 stimulation, the cytotoxicity of the CD4+T cell of AT-2SIV pulse.
Figure 11 a: with use oral LP(n=4) or iSIV(n=3) pretreated animal compare, by the extracorporeal antivirus effect active (in cd4 cell) of the autologous homology CD8+T cell that obtains with pretreated 8 Rhesus Macacus of oral iSIV/LP.
Figure 11 b: the extracorporeal antivirus effect active (in cd4 cell) of the xenogenesis (heterologous) of 4 acquisitions from oral iSIV/LP processes in 80 days 8 Rhesus Macacus or allogeneic (allogenic) CD8+T cell.
Figure 11 c-g: after oral immunity, the anti-SIV of 60 days CD8+T cells is active, at (c) that delay, insert in the culture systems of (d), allogeneic (e), when anti-MHC-la/ABC or anti-MHC-lb/E antibody (f) exist and removing TCR γ δ
+or V β 8
+in the CD8+T cell of subgroup (g).
Figure 12 a: with oral LP or the pretreated animal of iSIV, compare, with after internal rectum in the pretreated Rhesus Macacus of oral iSIV/LP and intravenous SIVmac239 challenge, the virus load level of blood plasma (the SIV RNA copy of every ml blood plasma).
Figure 12 b: with oral LP or the pretreated animal of iSIV, compare, with after internal rectum in the pretreated Rhesus Macacus of oral iSIV/LP and intravenous SIVmac239 challenge, the virus load level of cell (the SIV DNA copies of every 1,000,000 PBMC).
Figure 13: by the anti-CD8 antibody of infusion cMT807, remove peripheral blood and the lymph node CD8 of the macaque of 8 iSIV/LP processing
+t cell.A, peripheral blood CD8 before accepting three cMT807 injections and afterwards
+t cell counting; B, lymph node CD8 before accepting three cMT807 injections and afterwards
+t cell %; C, plasma viral load before accepting three cMT807 injections and afterwards; D, the PMBC DNA SIV carrying capacity after before accepting three cMT807 injections; E, lymph node SIV DNA carrying capacity before accepting three cMT807 injections and afterwards.
Figure 14: 8 Rhesus Macacus of using the oral formulations immunity of being made by iSIV and LP and 2 other untreated (
) monkey in SIVB670 internal rectum, carry out for the third time after internal rectum challenge, blood plasma (a) and virus load PBMC(b).
Figure 15: with (iSIV/LP immunity No.2), in vitro and in vivo CD8 after iSIV and LP Intragastric immunization
+the antiviral activity that T is cell-mediated.A, by 8 Rhesus Macacus of being challenged by internal rectum, during the 60-420 after immunity days, CD8
+the anti-SIV active (multiple that virus suppresses) of T cell; B and c, with those 8 Rhesus Macacus of oral iSIV/LP immunity and only use LP(n=4) or only use iSIV(n=4) in 8 contrast monkeys of processing, after internal rectum SIVmac239 challenge, the virus load of blood plasma and cell.
Figure 16: in 8 macaques (iSIV/LP immunity No.2), after the challenge of SIVmac239 internal rectum, in mucous membrane of rectum intraepithelial lymphocyte (IPL) (a-b), in lamina propria (lamina propria) cell (LPC) (c-d) and pelvic lymph node in (PLN) SIV DNA and RNA carrying capacity (e).
The specific embodiment
The detailed description of invention
The pharmaceutical composition of the mixture that the present invention relates to comprise antigen and non-pathogenic antibacterial alive.
Antigen
Variability due to sudden change in HIV genome, restructuring, insertion and/or due to lacking, HIV is classified as group, subgroup, type, hypotype and genotype.Have two main HIV groups (HIV-1 and HIV-2) and many subgroups, reason is that HIV genome constantly suddenlys change.Main difference between group and subgroup is relevant to peplos (envelope).HIV-1 is classified as main subgroup (M), and described subgroup M is classified as called after A to 9 hypotypes (branch (clade) or hypotype) (people such as Hu, JAMA275:210-216,1996 of J; The people such as Korber, Science280:1868-1871,1998), and the subgroup (O) outside the 10th.Also there are many other subgroups people such as (, Virus Genes2003,26:151-163) Papathanasopoulos MA come from restructuring in previous body.Preferably, HIV virus is HIV-1 or HIV-2, comprises all known and unknown its branches so far.Yet, HIV-1 preferably.
In the context of the present invention, " antigen ", from HIV source, this means that it is relevant to the combination of specificity HIV group, subgroup, type, hypotype or some hypotypes.Preferably, described HIV antigen is HIV-1 or HIV-2 antigen.
Described antigen right and wrong are infective.
CD4 suspects in scientific circles for a long time
+the activation of T cell (being the main target spot of HIV-1 and SIV) directly causes virus replication (Andrieu and Lu, 1995; Korin and Zack, 1999).But, only recently, just clarified CD4
+interaction between the consecutive steps of t cell activation and SIV or HIV course of infection.At static CD4
+in T cell, after entering in 2 hours, following virus after penetrating is Gag and the Pol albumen epi-position of offering on plasma membrane from the virion entering, and Env and Nef protein requirement synthetic people such as (, 2007) Sacha again.But the course of infection stage subsequently, that is, the reverse transcription after viral integrase develops very inefficently people such as (, 2009a and 2009b) Vatakis in static cell.On the contrary, within 48 hours when offer Gag and Pol epi-position on plasma membrane before or after, activate CD4
+t cell, HIV/SIV reverse transcription and DNA integrate activation completely, and it allows very effective virus replication and release people such as (, 2009a and 2009b) Vatakis.
Thereby, inventor's supposition, after HIV/SIV exposes, early stage HIV/SIV Gag or the Pol specific C D4 occurring of body internal specific blocking-up
+the activation of T cell, will cause preventing the virus replication of activation.
Consider this point, in order to induce HIV Gag and/or Pol angtigen presentation CD4
+the inhibition of t cell activation, prevents that in body, HIV copies and propagates in the people who then exposes in virus, and pharmaceutical composition of the present invention comprises HIV antigen, and it preferably has one or more epi-positions from HIV Gag and/or Pol albumen.Such antigen advantageously or contain or derived from HIV Gag and/or Pol.
Thereby term " contain or derived from the Gag of HIV virus and/or the antigen of Pol " refers to HIV antigen:
-comprise at least one Gag and/or Pol(as " antigen that contains Gag and/or Pol "); Or
-comprise one or more by GAG, for example capsid protein (p24) and stromatin (p17), the albumen of coding, and/or one or more by POL, for example intergrase, reverse transcriptase and protease, the albumen of coding (as " derived from the antigen of Gag and/or Pol "); Or
-comprise one or more epi-positions from these albumen (also as " derived from the antigen of Gag and/or Pol ").
Especially, being selected from following any other virus protein or its epi-position is not the necessary component that is contained in disclosed pharmaceutical composition in literary composition: the VPU of ENV, VIF, VPR, HIV-1, the VPX of HIV-2, REV, NEF, TAT etc.If existed, any of these albumen is only used for the optional components in disclosed pharmaceutical composition in literary composition.
Preferred antigens is particulate antigen.This means recombinant bacteria or fungus that it is preferably selected from virion, recombinant virus particle, virus-like particle, expression Gag and/or Pol, on its surface, offer one or more virus proteins or peptide or epi-position the polymerization microparticle of (contain or derived from HIV Gag and/or Pol).Preferably, one or more epi-positions from Gag and/or Pol produce or express or be contained in described antigen by described antigen.When using recombinant virus particle or virion or expressing the recombinant bacteria of Gag and/or Pol or during fungus, it is the microorganism of inactivation preferably.
Antigen can be virion, recombinant virus particle, virus-like particle or recombinant bacteria or the fungus of expressing Gag and/or Pol.It can also be one or more virus proteins or peptide (contain or derived from Gag and/or the Pol of HIV), restructuring or nonrecombinant, can be the form of conjugate (conjugate) or concatemer (concatemer).Then, antigen is the non-dependence of viral nucleic acid, that is, it is that non-viral DNA or non-viral RNA rely on.
Antigen can be from the expression that is advantageously contained in the nucleic acid sequence in suitable recombinant microorganism.
If the antigen being contained in pharmaceutical composition of the present invention is the recombinant bacteria of expressing Gag and/or Pol, so described recombinant bacteria is preferably different from the non-pathogenic antibacterial alive being also contained in compositions.
When the antigen in pharmaceutical composition according to the present invention is one or more virus proteins or peptide (contain or derived from Gag and/or the Pol of HIV), it is the form of granule preferably.In practice, suitable particulate antigen, can be by the microorganism of living (for example, yeast), in the mode identical with recombinant DNA hepatitis B vaccine, produce, in the situation of recombinant DNA hepatitis B vaccine, the HBsAg polypeptide self assembly of expression becomes immunogenicity spheroidal particle, the natural 22-nm granule of finding in its very similar chronic HBV infection patients serum (people such as Plotkin, 2008).
Or when the antigen in pharmaceutical composition according to the present invention is one or more virus proteins or peptide (contain or derived from HIV Gag and/or Pol), it is the form of conjugate.In such embodiment, as known in the art, interested albumen or peptide covalency are puted together in suitable carrier.The conventional carrier can business obtaining is albumen especially, as KLH(keyhole limpet hemocyanin) albumen, BSA(bovine serum albumin) albumen, OVA(ovalbumin) albumen etc. (preferably its can safely by oral administration to the mankind).Producing the method for suitable conjugate is familiar with those skilled in the art.
But, or when the antigen in pharmaceutical composition according to the present invention is one or more virus proteins or peptide (contain or derived from HIV Gag and/or Pol), it is the form of concatemer.As known in the art, concatemer is formed by a plurality of copies of proteins of interest or peptide, and in a macromole, physical connection is together for it.In concatemer, a copy of proteins of interest or peptide can directly or be synthesized arm separation and be connected to other copy.Thereby concatemer comprises at least two copies, preferred 10 copies or more of as many as, proteins of interest or peptide.The method of producing suitable concatemer belongs to those skilled in the art's common sense.
As used in the text, " virus-like particle " (VLP) refers to the granule of very similar mature virion, but it does not contain the viral genome material of described virus.More accurately, VLP, also referred to as pseudovirion, the subunit structure that representative is comprised of a plurality of copies of viral capsid and/or other virus protein.These virus proteins in vivo self assembly become the VLP of clear and definite spherical symmetric.These VLP do not comprise any nucleic acid molecules of the virus protein of encoding, and more accurately, it does not comprise any nucleic acid molecules.Thereby VLP is non-copying and non-infection in nature, this uses it safely with the form of pharmaceutical composition.For the production of the method for VLP be well known to a person skilled in the art (referring to, for example, the people such as Liew, 2010; Plummer and Manchester, 2010).For the production of the unrestriced example of the proper method of VLP at US5,919,458, EP386882, WO91/07425, US5,861,282 and WO91/05864 in described, it discloses and has not comprised the HIV VLP(pseudovirion that HIV genome does not comprise any nucleic acid molecules yet).
As described herein, " recombinant virus particle " refers to comprise from the albumen of different virus or exposes the virion from the albumen of different virus on its surface.And recombinant virus particle can also refer to antibacterial or other host cell, it comprises, produces or on its surface, exposes one or more contain or derived from virus protein or peptide or the epi-position of HIV Gag and/or Pol.
In fact, the great majority of recombinant virus particle are the virions that the part of wherein prototype structure albumen (that is, main envelope protein and core protein) is replaced by the correspondence from another virus (counterpart) albumen.As an example, envelope protein is interchangeable.In such a case, recombinant virus particle comprises " chimeric " genome being present in viral genome, and it has the sequence of the encoded packets memebrane protein exchanging from the sequence of another viral envelope protein with coding.Most of recombinant virus particles are reproducible and have infective.
As used herein, the recombinant virus comprising from another viral albumen refers to, inner or be presented on its surface, contains one or more contain or derived from the virus protein of HIV Gag and/or Pol or the recombinant virus particle of peptide or epi-position.Unrestricted example for the production of the method for recombinant virus particle has been described:
* restructuring Alphavirus Alphavirus (Alphavirus): in WO02/053757, disclose expression HIV(ENV albumen).
* retrovirus: in EP1499736, disclose slow virus (lentiviral) carrier of expressing chimeric glycoprotein albumen.
* adenovirus (for example 5,7 or 35 types): in US2007/077257, US2007/054395, JP2007037402, WO2006/120034, US2004/253210, US2004/170647, US2005/070017, US2003/228329, US2004/101957, US2003/219458, US2004/009936, US2004/028652, WO03/050238, WO03/038057, WO03/020893, WO02/31168, WO02/22080, WO01/02607 and US6716823, disclose the recombinant adenovirus of expressing HIV albumen.
* poxvirus (canary pox (canarypox), cattle pox, Ankara cowpox and fowlpox virus): US5, 766, 598, EP0592546, US2007/048861, US2006/188961, US2006/134133, EP1789438, WO2005/017208, WO2004/035006, US2004/146528, JP2003321391, EP1378516, WO95/07099, JP7170982, DE4141741, EP0449116, JP1148183, JP1085072, EP0592546, EP0243029, US2005/287162, JP2004105187, JP2004089185, WO03/095656, EP0592546, WO96/40880, US6, 136, 318, US5, 670, in 367, it discloses the recombinant poxvirus of expressing the virus protein that comprises HIV albumen.
* contain, produce or expose at least one antibacterial from viral albumen: US7 on its surface, 189,402 and WO96/11708 in, it discloses Salmonella (Salmonella) or the escherichia coli (E.coli) of expression HIV glycoprotein (that is, envelope protein).
Preferably, recombinant virus particle is corresponding to poxvirus, this poxvirus (is for example preferably selected from canary pox, ALVAC viral vector, as be described in patent US5,766,598 and EP0592546 in), cowpox (vaccinia virus for example, as be disclosed in International Patent Application WO 95/07099), Ankara cowpox (as, NYVAC viral vector, as be disclosed in patent application EP1789438) and fowlpox virus (as, TROVAC viral vector, as be disclosed in International Patent Application WO 03/095656).
More preferably, described poxvirus is canary pox virus.An example as the recombinant virus particle corresponding to canary pox virus and expression HIV peptide/albumen, can quote and be disclosed in patent US5, 766, 598(walks to the 82nd hurdle the 36th row from the 6th hurdle the 18th and is incorporated herein for referencial use) ALVAC viral vector, wherein this ALVAC carrier is expressed HIV-1gp120 as an example, HIV-1gp160, the secreted form of the non-cutting of HIV-1env, HIV-1gp120 with the grappling of cross-film sequence, HIV-1gag/pol, HIV-1gag/pol and env (gp120), HIV-1gag/pol and env (gp160), and HIV-1gag/pol and env(are with the gp120 of cross-film grappling).Preferably, described ALVAC vector expression HIV-1gag/pol and env (gp120), most preferably described ALVAC carrier is ALVAC vCP1521.
" virion " be SIV or HIV granule preferably, as the viral genome that contains sudden change (for example,, by nucleic acid mutation, replacement or insertion) causes producing SIV or the HIV virion of the virion of non-infection.
The virus genomic virion that comprises sudden change is disclosed in US7, and 229,625, US6,121,021, US6,923,970, US6,544,527, US6,451,322 and US6,080,408.
Advantageously, in order to make virion or recombinant virus particle safely use in people, described virion or recombinant virus particle are inactivations before using.Such inactivation, to recombinant virus particle, even to non-those that copy, may be necessary.
" virion of inactivation " as used in the text, described virion is restructuring or nonrecombinant, refers to no longer to infect the virion preferably no longer copying.
Method for inactivation virion or recombinant virus particle is known to those skilled in the art.Virally inactivated unrestriced example comprise chemical inactivation for example formalin, taurine chloramines, formaldehyde, paraformaldehyde, propiolactone, β propiolactone (REMUNE) or aldrithiol-2(without translation in correspondence) (AT-2; referring to US6; 001,155) processing, heat inactivation, physics inactivation are as U.V or gamma-radiation or Microwave exposure and combination thereof.For the reference of HIV inactivation, referring to the people such as RAVIV (J.Virol., vol.79 (19), p:12394-12400,2005).
According to an embodiment, described inactivation is chemical inactivation, and it is selected from formalin, taurine chloramines, formaldehyde, paraformaldehyde, propiolactone, β propiolactone (REMUNE) or aldrithiol-2 inactivation.
Or, or extraly, described inactivation is heat inactivation.Such inactivation is that technical staff is known, as the example of the method, can quote in embodiment disclosed.In fact, inventor in macaque, set up chemically astoundingly (that is, and AT-2) and/or the virus of heat inactivation, when combining with non-pathogenic activity bacterium, its induction protective immunity tolerance.
Advantageously, in order to be applied to people's object, virion is inactivation twice at least, typically uses at least above-mentioned two kinds of method for deactivating.
Preferably, as mentioned above, virion (restructuring or nonrecombinant as the antigen in pharmaceutical composition of the present invention, VLP or non-VLP) be nucleic acid (, DNA or RNA) non-dependence, this means that virion does not contain any viral DNA or RNA, if or its contain DNA or RNA, it is not effect in immunogenicity.
Or, the polymerization microparticle of various structures (with the form of microcapsule, microsphere etc.) and offer on its surface one or morely contain or derived from virus protein or peptide or the epi-position of HIV Gag and/or Pol, can be used as according to the antigen in pharmaceutical composition of the present invention.Such microparticle can be by the polymer formation of suitable biological or chemical, for example, methacrylic acid glucosan, methacrylic acid macrogol ester and/or gelatin, contain or can be adhered thereto derived from the HIV virus of HIV Gag and/or Pol or virus protein or peptide or epi-position.The example of polymerization microparticle is found in document (such as people such as Wei Li Lee, (2010), the people such as Sandri (2007), the people such as Goldberg (2003), Delie F. (1998), the people such as Ponchel (1998), the people such as Mathiowitz (1997), the people such as Fasano (1997), the people such as Chickering (1997)).
One preferred embodiment, according to the antigen in HIV-1 pharmaceutical composition of the present invention, be to express one or more contain or derived from the virus protein of HIV-1Gag and/or Pol or one or more virions of peptide or epi-position.Or, according to the antigen in HIV-1 pharmaceutical composition of the present invention, be on its surface, to offer one or more contain or derived from the virus protein of HIV-1Gag and/or Pol or one or more polymerization microparticles of peptide or epi-position.
Preferably, for according to the antigen of pharmaceutical composition of the present invention being at least about 110kDa size.Preferably, at least about 120,130,140,150,160,170,180,190,200kDa size is even larger.
Use conventional general knowledge, and on the basis of the embodiment of following discloses, in conjunction with SIV or HIV virus, technical staff is easy to be identified for the effective dose of the virus antigen in the context of the invention.
As an example, when described antigen is particulate antigen and more particularly during virion, the amount of virion is about 10
6to about 10
12mixture described in every mL.
Non-pathogenic bacteria
As the inventor uses as shown in the SIV in macaque, when by mucosa or Intradermal or intraepithelial approach when using together with suitable as defined above antigen, the state to the immunologic tolerance of above-mentioned antigen can be induced and preferably be maintained to the non-pathogenic antibacterial alive being included in pharmaceutical composition.In people, this makes to prevent and/or treat HIV disease.
Thereby, described antibacterial can be considered to specific adjuvant, it can be designated as " adjuvant that causes tolerance " or " causing tolerance carrier (carrier) " or " causing tolerance carrier (vehicle) " or " toleration carrier " or " tolerance effect (tolerization) carrier " or " for the carrier tolerating " in the text, and these terms are synonyms.
Preferably, all these terms that are equal to refer to, in order to obtain the specific immunity protection (preferably, immunologic tolerance) to antigen, thereby prevent and/or treat the HIV disease in people, with the non-pathogenic antibacterial alive that HIV antigen is used in combination as defined above.
More preferably, " cause tolerance carrier " is non-pathogenic antibacterial alive, and it mixes and use with HIV antigen as defined above, with realize following immunoprotection effect one or more, preferably 2 or a plurality of, more preferably 3 or a plurality of:
1) " cause and tolerate carrier " the remarkable generation of not inducing whole body HIV antigen-specific antibodies:
Especially, do not observe the remarkable generation of the anti-HIV IgM of whole body and/or IgG antibody.For example, there is no significant whole body humoral response, by classical clinical laboratory method, the detectable whole body antibody response of specificity do not detected as ELISA in other words, if or whole body antibody detected, for anti HIV-1 virus, infecting neither protectiveness.
2) " cause tolerance carrier " and do not induce significant HIV antigenic specificity CD4+T cell proliferation:
Especially, by the standard analysis of describing in appended embodiment for example, measure, during HIV antigenic stimulus, do not observe significant HIV antigenic specificity cd4 cell propagation in vitro.
3), while " causing and tolerating carrier " HIV antigenic stimulus in vitro, do not induce the IFN-γ being produced by CD8+T cell significantly:
Especially, in vitro during HIV antigenic stimulus, below the threshold level that the IFN-γ level by CD8+T emiocytosis of observing is analyzed at ELIspot.
4) induction suppresses the remarkable CD8+T cell effect of HIV angtigen presentation CD4+T cell-stimulating " to cause tolerance carrier ":
Especially, as enclose as shown in embodiment, can determine this reaction by measuring the testing in vitro of the virus replication level (indication " significantly " CD8+T cell effect) being suppressed by CD8+T cell.These CD8+T cells are also called CD8+ " adjusting " T cell.But, especially, consider, for example the remarkable generation of IFN-γ is not induced in this reaction, and these reaction right and wrong are Cytotoxic.But especially, this reaction is MHC-1b/E restriction.But especially, TCR α β seems to participate in suppressing the CD8+T cell effect of virus replication.But especially, with the same cell faciation ratio of removing CD8+T cell, this reaction suppresses the activation of HIV angtigen presentation CD4+T cell.Preferably, described reaction suppresses the early stage activation of HIV angtigen presentation CD4+T cell, and wherein said " in early days " activates and measure (Scholzen and Gerdes.J.Cell Physiol182,311-322 (2000 March)) by Ki67+ label.
By above 1), 2) and 3) in the term that uses " do not induce ", mean for suitable quantitative detecting analysis, lower than the result of threshold level, wherein said " threshold level " is according to negative control definite value in analysis: at this, below value, result is negative findings.This value can be different and different according to analyzing, and detection method is different and different.
Advantageously, causing tolerance carrier is selected from alive:
-non-pathogenic bacteria, particularly probiotic bacteria (probiotics) and symbiotic bacteria;
The malignant bacteria of-attenuation; With
-inactivation (alternatively, be also above-mentioned attenuation) malignant bacteria.
Causing tolerance carrier can be restructuring or nonrecombinant.
As " non-pathogenic bacteria " that cause tolerance carrier, conventionally in people, do not induce any pathology in the context of the present invention.This is also its reason that is conventionally considered to safety (GRAS).Certainly, such antibacterial should be applied to people.
As the preferred non-pathogenic bacteria that causes tolerance carrier, it is symbiotic bacteria.Such antibacterial is that technical staff is known.Unrestriced example comprises bacillus (Bacillus sp) (for example, bud bubble bacillus B.coagulans), animal bifidobacteria (Bifidobacterium animalis), bifidobacterium breve (Bifidobacterium breve), bifidobacteria infantis (Bifidobacterium infantis), bifidobacterium longum (Bifidobacterium longum), bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium lactis (Bifidobacterium lactis), escherichia coli (Escherichia coli), bacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), lactobacillus casei (Lactobacillus casei), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus salivarius (Lactobacillus salivarius), lactococcus lactis (Lactococcus lactis), streptococcus thermophilus (Streptococcus thermophilus) etc.
As " symbiotic bacteria " that causing tolerance carrier in the context of the invention advantageously lactobacillus or bacillus bifidus, it is more particularly selected from above list, also comprises its combination.A preferred symbiotic bacteria is Lactobacillus (Lactobacillus sp.), and Lactobacillus plantarum more preferably.Below the embodiment of report shows that Lactobacillus plantarum is to cause tolerance carrier for the first time, when when antigen is together used as defined above, causes virus immunity tolerance.
Advantageously, the combination of non-pathogenic bacteria, as two or more symbiotic bacterias, can be used as causing tolerance carrier.
As used in the text, term " malignant bacteria " refers to induce the antibacterial of pathology in people.Such antibacterial is known to technical staff, comprises especially listeria (Listeria species) (for example Listeria Monocytogenes (Listeria monocytogenes)), corynebacterium (Corynebacterium species), Mycobacterium (Mycobacterium species), Rhod (Rhococcus species), Eubacterium (Eubacteria species), Bordetella (Bortadella species) and Nocardia (Nocardia species).Preferably, malignant bacteria is selected from Mycobacterium (Mycobacterium species), more preferably Mycobacterium bovis (Mycobacterium bovis).
As used in the text, " attenuation malignant bacteria " be due to one or more sudden changes or one or more attenuation treatment (for example, chemical treatment and/or the continuous passage on particular medium), compare the malignant bacteria that toxicity (virulent) is lower with its wild type counterparts.This attenuation malignant bacteria is well known to a person skilled in the art.The unrestriced example of attenuation malignant bacteria comprises the mycobacteria of the Salmonella typhimurium (Salmonella typhimurium) of attenuation and the mycobacteria of preferred attenuation.Example as attenuation mycobacteria, can quote " bacillus calmette-guerin vaccine (Bacille de Calmette Guerin) ", be also referred to as " BCG ", and, more particularly, wherein, two strains in late period (BCG Danish and Glaxo) of evolving in the BCG strain of six extensive uses-early stage strain BCG Japanese, the DU2 group of evolving III, and three strains in late period (BCG Connaught, Pasteur and Tice) of evolving in DU2 group IV.Other example as attenuation mycobacteria, can also quote recombinant BCG, as be disclosed in the strain rBCG30 in the people such as HOFT (2008), be disclosed in the recombinant BCG in the people such as WANG (2008), and be disclosed in International Patent Application WO 2005/111205 and WO02/102409 and be disclosed in patent US7,122,195 and US6, recombinant BCG in 261,568.
Advantageously, be not attenuation or when attenuation, malignant bacteria can by inactivation with as the context of the invention cause tolerance carrier, still, attenuation malignant bacteria also can be used after by inactivation.
" inactivation malignant bacteria " is well known to a person skilled in the art.The preparation method of this inactivation malignant bacteria forms a part for the conventional general knowledge in this area.Example as this method, can enumerate the cracking of phage mediation, chemical inactivation is processed and (is seen US7 as formalin, 393,541), heat inactivation, as lyophilization (for example, and their combination expansion lyophilization) or the physics inactivation of U.V or gamma-radiation (referring to WO2008/128065) or Microwave exposure.
Preferably, described in, causing tolerance carrier is that malignant bacteria is as the attenuation derivant of BCG.Below the example of report shows that BCG causes tolerance carrier for the first time, when causing viral immunologic tolerance when antigen is together used as defined above.
When restructuring, the tolerance carrier that causes according to the present invention is not expressed any HIV albumen or peptide or epi-position.
Preferably, the significant quantity that is at least used as the antibacterial alive that causes tolerance carrier is at mid-log phase.More preferably, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even about 100% bacterial cell sum is at mid-log phase.
Technical staff can easily determine the effective dose that causes tolerance carrier, and the example of this effective dose is in following discloses.
As an example, the amount of antibacterial described in pharmaceutical composition of the present invention is about 10
4to about 10
14mixture described in the every ml of CFU.
Pharmaceutical composition
In described compositions literary composition, also as " causing tolerance compositions " or " tolerance-immunogenic composition ", these terms are equal to.
Cause tolerance carrier and contain or derived from the Gag of HIV virus and/or the antigen of Pol be two minutes other, different component, it is included in pharmaceutical composition of the present invention as mixture.Described in this means, causing tolerance carrier and described antigen is present in described compositions as different components.
Advantageously, pharmaceutical composition of the present invention does not comprise any oligonucleotide (for example, CpG or dsRNA) as adjuvant.
Owing to causing tolerance carrier, be antibacterial, the antibacterial of same genus and/or kind can be used separately as the source of antigen under recombinant forms.For example, recombinant bacteria is positioned at suitable adjusting sequence and (comprises promoter containing, derivable or composition) nucleic acid of coding for antigens under control, this nucleic acid can be contained on intracellular nucleic acid carrier, or is integrated into bacterial chromosome as the nucleotide sequence of integrating.Thereby described antigen can be expressed or produce to recombinant bacteria.Thereby, according to specific embodiment, pharmaceutical composition of the present invention comprise be antibacterial cause tolerance carrier, and be one or more contain or derived from the virus protein of HIV Gag and/or Pol or the antigen of peptide or epi-position, and its dividually by with cause that tolerance carrier belongs to identical genus and/or the recombinant bacteria of kind produces.
In pharmaceutical composition according to the present invention, when described antigen is particulate antigen and more particularly during virion, at virion described in described mixture (being expressed as the granule in mixture described in every ml), to the ratio of described antibacterial (be expressed as described in every ml in mixture CFU), be about from 1:10 to about 1:1000, preferably from about 1:25 to about 1:750, yet preferably from about 1:50 to about 1:500, even more preferably from about 1:75 to about 1:250, yet be more preferably about 1:100.
Use pharmaceutical composition of the present invention
Likely side by side or dividually or one after the other use and cause tolerance carrier and antigen.
Thereby, an object of the present invention is to provide for preventing and/or treating the pharmaceutical kit of the people HIV disease needing, it contains:
-in the first container, antigen as defined above; With
-in second container, non-pathogenic antibacterial alive as defined above,
Wherein said antigen and described antibacterial for mucosa or Intradermal or on the pharmaceutically acceptable carrier of intradermal administration.
Another object of the present invention is to provide product, and product contains:
-as defined above as the non-pathogenic antibacterial alive that causes tolerance carrier; With
-there is as defined above particulate antigen or the antigen of one or more epi-positions from HIV Gag and/or Pol albumen,
As for composition of medicine compositions simultaneously, that separate or that in succession use, for preventing and/or treating the people HIV disease needing.By mucosa ground or Intradermal ground or upper Intradermal to described people, use described combination pharmaceutical composition prevent and/or treat described in realizing.For this object, can be side by side or use dividually or one after the other and cause tolerance carrier and antigen.
As an example, non-pathogenic antibacterial alive can oral (for example, as oral drugs or dietary supplement ingredient), and antigen be mucosa ground or Intradermal ground or upper Intradermal use.
Certainly, for example, in order to guarantee each suitable expection site (, mucomembranous surface) that is delivered to, can use suitable pharmaceutical carrier.Technical staff uses by adjusting easily time and the dosage that respectively causes tolerance carrier and antigen.
Preferably, pharmaceutical composition according to the present invention is mucosa or Intradermal or intraepithelial pharmaceutical composition.But preferably, it is oral pharmaceutical composition.
As used in the text, " mucosa or Intradermal or intraepithelial pharmaceutical composition " is the pharmaceutical composition for mucosa or Intradermal or upper intradermal administration, this means that it is formulated for such using.
Especially, pharmaceutical composition can further comprise for mucosa or Intradermal or upper Intradermal and sends the one or more suitable pharmaceutical carrier (or holder) of described antigen and described antibacterial.
Preferably, " mucosal delivery " in literary composition is selected from nose, mouth, Sublingual, trachea, pharynx, bronchus, esophagus, Stomach duodenum, small intestinal, rectum, foreskin and vagina and sends." mucosal delivery " is to be delivered to mucomembranous surface, as the mucosa of nose, mouth, Sublingual, trachea, bronchus, pharynx, esophagus, stomach and duodenum, little and large intestine, comprise mucous membrane of rectum, and foreskin and vaginal mucosa.In the present context, mucomembranous surface also comprises the outer surface of eyes, that is, and and eyes and mucosa around thereof.Yet preferably, mucomembranous surface refers to vagina and gastrointestinal mucosal, more preferably gastrointestinal mucosal.Yet preferably, mucosal delivery is oral delivery.
Thereby pharmaceutical composition can also comprise one or more pharmaceutical carriers that depend on route of administration.The those of ordinary skill of pharmaceutical field is known, or can easily be identified for drug delivery to the carrier of mucomembranous surface, or the carrier of sending for Intradermal or upper Intradermal.About this respect; useful reference is Chien(Novel Drug delivery system; chapter 3, from 6 to 9; Marcel Dekker; 1992), Ullmann ' s Encyclopedia of Industrial Chemistry, sixth version (multidigit editor; 1989-1998, Marcel Dekker); And Pharmaceutical Dosage Forms and Drug Delivery Systems (people such as ANSEL, 1994, WILLIAMS & WILKINS).
For the illustrative methods of drug delivery useful in the present invention and approach in following summary.
Can for example, by pharmaceutical composition being mixed with to suction, spraying etc. (, spray nose, arosol spray or pump spraying etc.), solution, gel etc., obtain to using of bronchus, bronchioles, trachea, nose, mouth, foreskin or pharyngeal mucous membrane.The sprayer device that is applicable to the pharmaceutical composition to be delivered to nasal mucosa, trachea and bronchus is known in the art, therefore here will not be elaborated.So, pharmaceutical composition can comprise the carrier of the emulsion that is selected from solution, emulsion, microemulsion, emulsion oil-in-water, anhydrous lipid and emulsion oil-in-water and other type.
Can obtain by pharmaceutical composition being mixed with to solution, enema, foam, suppository, vaginal tablet or topical gel to using of vaginal mucosa.Being preferred for carrier that vagina sends comprises and hydrophilic and hydrophobic carrier is for example commonly used in those (for example, oil/water emulsion gels) in preparation emulsion or gel preparation.
To using of gastrointestinal mucosal can be by pharmaceutical composition be mixed with to capsule, microcapsule obtains.The preferred vector correspondence of sending for digestive tract (for example common peroral administration capsule and microcapsule, the capsule of pectin and/or alginate and microcapsule), as being generally used for, prepare digestive tract delivery formulation those (for example, being disclosed in the microcapsule in International Patent Application WO 2007/140613).Or, can be by digesting or using suitable liquid and/or food, the digestive tract such as the acquisition such as beverage, Yoghourt is sent.
Intradermal or upper intradermal administration are that technical staff is known.For example can use needle device to carry out intradermal administration (for example, injection), patent US6 for example, 933,319 and International Patent Application WO 2004/101025 in disclosed, or undertaken by suitable needleless device.
Pharmaceutical composition can further comprise at least one absorbent." absorbent " is well known to those skilled in the art.For example, the surfactant of can giving an example, for example polyoxyethylene deriv of sorbitol anhydride fatty acid partial ester (for example, tween
80, s6, polyoxyethylene 50 stearate, laureth9 and Octoxinol), bile salts is as NaGC, mixed micelle, enamine, nitric oxide donors (as, S-nitrosoglutathione-N-acetyl group-DL-penicillamine, NOR1, NOR4-its preferably with NO scavenger as carboxyl PITO or diclofenac sodium co-administered), sodium salicylate, the glyceride of acetoacetic acid (for example, glyceryl-1,3-diacetyl acetic ester or 1,2 – isopropylidene glycerol-3-acetoacetic ester), cyclodextrin or beta-cyclodextrin derivative (for example, 2-HP-BETA-CD and seven (2,6-, bis--O-first group-beta-cyclodextrin)), medium-chain fatty acid, as single-and two glyceride (for example, the Capric acid sodium salt extract of Oleum Cocois, Capmul), or triglyceride (as, amylodextrin, Estaram299, Miglyol810), polymer is as carboxymethyl cellulose, carbomer, polycarbophil, Tragacanth and sodium alginate, and other is suitable for mucosa or Intradermal or the intraepithelial absorbent of sending.About successfully for the reference of the rule of the absorbent of mucosa or Intradermal or upper Intradermal drug delivery, referring to Chien, Novel Drug Delivery Systems Ch.4(Marcel Dekker, 1992).
Pharmaceutical composition can further comprise one or more additives (for example, diluent, excipient, stabilizing agent, antiseptic etc.).Usually, referring to, Ullmann ' s Encyclopedia of Industrial Chemistry, sixth version (multidigit editor, 1989-1998, Marcel Dekker); With Pharmaceutical Dosage Forms and Drug Delivery Systems (people such as ANSEL, 1994, WILLIAMS & WILKINS).
As following discloses, by being applied to can determining according to one or more features of described experimenter according to the suitable dosage of pharmaceutical composition of the present invention of people experimenter, as sex, age, body weight, health etc.
As an example, when antigen is granule, more particularly, when it is virion, can to described people, use about 10 every day
8to about 10
14the dosage of individual virion.As another example, can to described people, use about 10 every day
6to about 10
16the dosage of the non-pathogenic antibacterial alive of CFU.
The application of pharmaceutical composition of the present invention
An object of the present invention is to provide pharmaceutical composition as above, as medicine, preferably as the purposes of vaccine.
The invention still further relates to for preventing and/or treating the method for the people HIV disease needing, comprise at least to described people's mucosa ground or Intradermal ground or upper Intradermal use the step of the pharmaceutical composition as defined above of effective dose.
According to the present invention, for the object of preventing, " having the people who needs " can be anyone, the preferred at least people of about 2 years old.For the object for the treatment of, " having the people who needs " is because he/her suffers from HIV disease by the people who is treated.
" HIV disease " refers to any immunological diseases relevant to HIV, comprises the commitment of AIDS and progression of disease, comprises seroconversion (chronically infected foundation).
The invention further relates to protection people and avoid the method for HIV, comprise at least to described people's mucosa ground or Intradermal ground or upper Intradermal use the step of the pharmaceutical composition as defined above of effective dose.
Especially, if mucosa ground is exposed to HIV, the method can be protected people to avoid HIV to infect, if and/or vein be exposed to HIV, the method can be protected people to avoid HIV and copy.
The present invention also relates to further protection people and avoids the method for HIV seroconversion, comprise at least to described people's mucosa ground or Intradermal ground or upper Intradermal use the step of the pharmaceutical composition as defined above of effective dose.Thereby described people will can not become seropositivity, can't show significant HIV antibody horizontal.
Term " inoculation " refers to the action that prevents and/or treats people HIV disease (especially, using pharmaceutical composition of the present invention) of taking.Preferably, pharmaceutical composition of the present invention for induction and, preferably, maintain people to containing or being useful derived from the immunologic tolerance of the Gag of HIV virus and/or the antigen of Pol, that is, in other words, to inoculation (or " causing tolerance ") described people, be useful.Thereby, use medicine inoculation people of the present invention to be considered to " causing tolerance inoculation " (or " tolerance effect " or " tolerance ").
If mucosa or Intradermal or on after intradermal administration pharmaceutical composition of the present invention (that is, after causing tolerance inoculation), in people, successfully induced immunologic tolerance, described people is considered to " being vaccinated " (or " by what tolerate " or " tolerance ").Body inner virus is infected to the reaction of challenge,, the virus replication of being assessed as the plasma viral RNA carrying capacity by " being vaccinated " people, with contrast people's (it has been used separately antigen or antigen with standard adjuvant (as defined above) or there is no drug administration compositions or placebo) plasma viral RNA carrying capacity and compare, reduced at least about 50%, more preferably at least about 70% more preferably at least about 75% or 80% or 85% or 90% or 95% or 98% or 99% yet, or even more (99.5%, 99.8%, 99.9%, 100%).
According to the present invention, cause the continuous administration that tolerance inoculation can comprise one or many pharmaceutical composition.Preferably, cause tolerance inoculation and can comprise at least twice or continuous administration (that is, inoculation) repeatedly, more preferably, compositions described in twice above continuous administration.
Advantageously, the interval causing continuously between tolerance inoculation is between 1 minute to 3 months, preferably between 15 minutes to 2 months.
But, advantageously, of the present inventionly cause tolerance inoculation and can also comprise for the first time mucosa or Intradermal or intraepithelially cause after tolerance inoculation 1 year or for many years, again cause tolerance inoculation (for example 1 to 10 year).
Mucosa or Intradermal or intraepithelial causing newly after tolerance inoculation cause tolerance inoculation and can be selected from mucosa, Intradermal and intraepithelially cause tolerance inoculation for the first time.Significantly, if new causing tolerates inoculation, be upper Intradermal or intradermal injection, specificity whole body body fluid and/or cell toxicant (generation IFN-γ) reaction is detectable so, but prevention of disease or treatment do not act on.
According to the present invention, the effective dose of pharmaceutical composition is applied to the people who needs.Term " effective dose " refers to be enough to realize the amount of required biological effect, and biological effect is herein by inducing immune tolerance, preferably healing or the protective effect (in other words, immune protective effect) of " Ts " immunologic tolerance.Can understand effective dose by depend on by the experimenter's who is treated age, sex, health and body weight, if any, simultaneously treatment kind, the frequency for the treatment of and the character of Expected Results.The scope of the effective dose below providing is not intended to restriction invention, represents preferred dosage range.But as understood by a person skilled in the art and can determine, capable of regulating preferred dose is to adapt to experimenter, this does not need excessively (undue) experiment.Referring to, for example, Ebadi, Pharmacology, Little, Brown and Co., Boston, Mass. (1985).
For example, about HIV, for adult's typical doses, be every dosage about 10
6-10
12hIV virion (that is, VLP, restructuring or non-recombinant virus granule), preferably 10
8-10
10.Certainly, no matter use how much dosage, it should be defined as by known method measuring safely and effectively, as described herein.
And those skilled in the art can also determine in order to realize required biological effect according to his/her general knowledge, will be applied to people's the effective dose that causes tolerance carrier.
As an example, the effective dose of the attenuation derivant of described malignant bacteria (for example BCG) is every dosage 10
4to 10
12scope, preferably 10
5to 10
10cFU(clones forming unit), more preferably 10
6to 10
8cFU.As other example, the effective dose of the attenuation derivant of the malignant bacteria of described malignant bacteria or inactivation (for example BCG) is the scope of every dosage 0.001mg to 1g, and preferably 0.01 to 100mg, and more preferably 0.1 to 10mg.
As other example, the effective dose of described non-pathogenic bacteria (for example Lactobacillus) is every dosage about 10
6-10
14the scope of CFU, more preferably about 10
10-10
12cFU.
As mentioned above, pharmaceutical composition of the present invention is suitable for preventing people's HIV disease in the future, or treats the people who has suffered from HIV disease.
For the object for the treatment of, " antigen, contain or derived from Gag and/or the Pol of HIV virus " as defined above, can be autologous, that is to say, it can be derived from infecting the people's who is treated HIV virus.In this case, for example, HIV virus can be separated from people, then, can cultivate virus and make its inactivation (preferably at least inactivation twice), thereby finally tolerate carrier and be combined and obtain pharmaceutical composition as above with causing.
Yet, for example, contain autologous homology or non-ly autologously contain or can during conventional antiviral therapy, be administered to people derived from the pharmaceutical composition of the antigen of HIV Gag and/or Pol, it is by the virus load that first causes can't detect.Then, as long as the suitably in vitro virus replication of having realized the cd4 cell of non-autologous homology actute infection by autologous homology virus specific C D8 cell suppresses, as long as or realized the suitable inhibition that the cd4 t cell of being induced by cd8 t cell activates, just at one or many, make causing after tolerance inoculation of pharmaceutical composition, stop conventional antiviral therapy.
Especially, for therapeutic purposes, pharmaceutical composition can only be used once being treated between people's vital stage.Or it can be used twice between people's vital stage or repeatedly being treated, on the same day or separate during certain not on the same day, the scope during described is for example from about 1 day to about 1 year, or longer.More particularly, it can every day or uses termly, during scope for example from about 1 day to about 1 year, or longer.If necessary, pharmaceutical composition can using the people who is treated throughout one's life.
The present invention further provides the method for the whether protected HIV of the avoiding virus of external definite people, having comprised:
A) separated peripheral blood cd8 t cell from the described blood sample that is vaccinated people;
B) cultivate under suitable condition:
(i) the cd8 t cell of described separation and allogeneic or autologous CD4+T cell, CD4+T cell is infected by the Strain being equal to described HIV virus in vitro acutely; With
(ii) the allogeneic of described external actute infection or autologous CD4+T cell;
C) reclaim culture supernatant;
D) measure the virus load in described supernatant; With
E) determine the whether protected described HIV virus of avoiding of described people.
By " Strain that is equal to HIV virus to be tested ", it means that described Strain comes from wild virus, and there is the substitutive characteristics similar to HIV to be tested virus (for example, can exemplify the Strain HTLVIIIB:HTLVIIIB that comes from individual HIV-1 and can be considered to " being equal to the Strain of HIV-1 ").Preferably, to be derived from be the wild virus of HIV virus to be tested to described Strain.Thereby described Strain has represented that research comprises HIV virus, the particularly suitable model of wild HIV virus.Certainly, Strain is adjusted to adapt to such research well, particularly aspect safety.
All steps can be used standard technique well known to those skilled in the art to carry out above.Particularly, the suitable condition of culture for step b) is a part (as the conventional method of describing in following examples) for general knowledge in field of the present invention.
By those conventional method energy measuring processs d as described in following examples) in virus load.
Virus load from the supernatant reclaiming according to the allogeneic of sub-step b) described external actute infection (ii) or autologous CD4+T cell culture is by the definite reference being used as in step e).By for example more only containing the geometrical mean from the virus concentration in the supernatant in the hole of duplicate (or in triplicate or in quadruplicate or more) of the allogeneic of external actute infection or autologous CD4+T cell, with contain cd8 t cell and from the geometrical mean of the virus concentration in the supernatant in the hole of duplicate (or in triplicate or in quadruplicate or more) of the allogeneic of external actute infection or autologous CD4+T cell, will advantageously calculate " suppressing percentage ratio (%) " or " suppression ratio " or " antiviral effect ".
So, described definite being preferably as follows in step e) carried out:
If-suppression ratio is higher than about 100, the described people that can reach a conclusion is protected.Typically; this will be following situation: if the people of the non-infection of HIV has been applied effective preventative antiviral therapy; if or the people that HIV infects has been applied effective therapeutic treatment; described effectively preventative or therapeutic treatment comprises preferably according to pharmaceutical composition of the present invention; thereby; as long as it is still protected, there is no need further to people, to use any preventative or therapeutic treatment.
If-suppression ratio is lower than about 100, the described people that can reach a conclusion does not have the protected described virus of avoiding.So, people, the people that the people of the non-infection of HIV or HIV infect; to advantageously be applied respectively the preventative or therapeutic treatment comprising according to pharmaceutical composition of the present invention, and, with reasonable time interval; by the in vitro method carrying out more than one or many, guarantee that people becomes protected.
And; the invention provides the test kit for the whether protected HIV of the avoiding virus of external definite people; it comprises allogeneic or the autologous CD4+T cell that can be infected by Strain, and described Strain as defined above, is equal to described HIV virus to be tested.Test kit can also contain enough Strain of debita spissitudo, to infect above-mentioned allogeneic or autologous CD4+T cell, and/or suitable reagent and/or contrast and/or medium (as the medium for cell suspension, cell culture, cell storage etc.).Test kit of the present invention can be specific to HIV virus ad hoc type, or it is adjustable to be applicable to viral multiple-type, and the type of described virus is (particularly, upper approaching occurs system) approaching.
Can easily adjust the present invention to be applicable to prevent and/or treat any chronic infectious diseases.The unrestriced example of such disease is: B and C type hepatitis, human papillomavirus (HPV), EBV and other herpesvirus, pulmonary tuberculosis, leprosy, leishmaniasis etc.
Globally, each time when one or several pathogenic antigens with above-mentioned infection or disease association participates in CD4+T cell (described CD4+T cell is offered the epi-position derived from above-mentioned pathogenic protein or peptide) specificity and activates, non-cell toxicity CD8+T cell (described CD8+T cell is by the drug regimen deposits yields that causes tolerance carrier described in the above-mentioned antigen of combination of mucosa or upper Intradermal or Intradermal and literary composition) can cause the specificity of CD4+T cell-stimulating is suppressed/prevents.
In literary composition, show to use pharmaceutical composition of the present invention can prevent and/or treat viral infection and the relevant disease in mammal/people.According to this instruction, other pharmaceutical composition is likely provided, it comprises: (i) as the disclosed tolerance carrier that causes in literary composition; (ii) any antigen from virus, antibacterial, fungus, protozoacide or parasite source.Such pharmaceutical composition is formulated for described in suitable sending (preferably, mucosa or Intradermal or upper Intradermal) and causes tolerance carrier and described antigen.Its for prevent and/or treat by antigen derived from the caused mammal of virus, antibacterial, fungus, protozoacide or parasite in chronic infection be useful.An example is antibacterial pharmaceutical composition, and it comprises the (i) as described herein tolerance carrier that causes; (ii) derived from the antigen of mycobacterium tuberculosis (Mycobacterium tuberculosis).This antibacterial pharmaceutical composition be formulated for the described antigen of suitable sending (preferably, mucosa or Intradermal or upper Intradermal) and described in cause tolerance carrier.Especially with interest, for what prevent and/or treat people's tuberculosis, be such antibacterial pharmaceutical composition, wherein antigen of mycobacterium is derived from KochShi bacillus.
The immunoprotection of realizing by pharmaceutical composition of the present invention
Tolerance is immune a kind of physiological potency, to identify the antigen entering by Mucosal system, and produces again meeting with the anergy (anergy) (usually relevant to other immune modification) of same antigen.Often show that tolerance brings out the antibody containment that the mucosal sIgA of mucosa antigen is allowed, and do not stimulate general immunity region.TGF-β, a kind of regulatory cell factor, also participates in the generation of tolerance sometimes.Also the activity that often shows CD25+ regulatory T cells suppresses potential mechanism (Faria and Weiner, 2005 as mucosal tolerance; The people such as Mestecky 2007).But, in the immunologic tolerance of the pharmaceutical composition induction of describing in the present invention, do not observe these immune modifications, be primarily characterized in that the CD8+T cytoactive of the activation of the CD4+T cell that suppresses to offer virus epitopes antigen, this immunoreation type is not recognized so far, more particularly, it is a kind of immunologic tolerance of brand-new type.
Statement " tolerance ", " immunology tolerance ", " immunologic tolerance ", " to viral immunologic tolerance ", " new virus specific tolerance ", " immunologic tolerance to virus antigen ", " immunologic tolerance former to virus immunity " and " " Ts " immunologic tolerance " are synonyms.Inventor has shown that this correspondence in macaque the CD8+T cell effect of the strong non-cell toxicity of active induction, MHC-1b/E restriction, it suppresses the early stage activation of HIV Gag and/or Pol angtigen presentation CD4+T cell, and relevant to the disappearance of CD4+T cell proliferation, and there is no the IFN-γ of CD8+T emiocytosis when the SIV of inactivation antigenic stimulus, and there is no the generation of the anti-SIV IgM of whole body and IgG antibody.And inventor can show that TCR γ δ and V β 8 do not participate in the CD8+T cell inhibition of virus replication, shown that TCR α β should play a major role in offering by MHC-1b/E peptide on the CD4+T cell of identification infection.
Especially, when containing or derived from the conventional preventative vaccine compositions of the Gag of HIV virus and/or the antigen of Pol and standard or conventional adjuvant (with comprising, object is stimulate and/or contribute to and/or any form of immunoreactive physics, chemistry or biological adjuvant that increase is relevant to antigen, as the people such as S.Plotkin describe in being entitled as " Adjuvants " chapter in " Vaccines " acceptance of the bid) during inoculation, can be observed " the common immunoreation to the antigen derived from viral ".Should " the common immunoreation to the antigen derived from viral " comprise immunoreation body fluid, cell or body fluid and cell, its common being characterized as:
(i) under specific in vitro stimulates, virus-specific cd4 cell is bred; And/or
(ii) by producing the whole body antibody induction specificity whole body humoral response of antiviral antigen protein and/or peptide; And/or
(iii) induction produces to cd8 t cell the specific cell reaction that IFN-γ is relevant, and/or
(iv) there is no CD8+T cell effect non-cell toxicity, that suppress HIV Gag and/or Pol angtigen presentation CD4+T cell-stimulating.
On the contrary, as provided by the invention, comprise and contain or derived from the Gag of HIV virus and/or the antigen of Pol with cause the drug regimen deposits yields " to viral immunologic tolerance " of tolerance carrier, and, more particularly, produce " " Ts " immunologic tolerance ", in below about macaque, in the embodiment of SIV, determined and it is characterized in that:
(i) under specific in vitro stimulates, there is no virus-specific cd4 cell propagation; With
(ii) without any significant whole body humoral response, that is to say, by classical clinical laboratory method, as ELISA, the detectable whole body antibody response of specificity do not detected, or if whole body antibody detected, it would not have protectiveness to SIV viral infection yet; With
(iii) under enough stimulated in vitro, the any cytotoxicity CD8+T cell effect relevant to producing IFN-γ is not (for example, detectable by ELIspot), if or the reaction of cytotoxicity cd8 t cell detected, it does not have protectiveness to SIV viral infection yet; With
(iv) as a key character, the CD8+T cell effect of the strong non-cell toxicity of active induction, non-CD25MHC-1b/E restriction, suppresses the early stage activation of SIV angtigen presentation CD4+T cell.
As mentioned above, pharmaceutical composition of the present invention comprises and causes tolerance carrier and antigen, and described antigen contains, or derived from Gag and/or the Pol of HIV virus.In fact, be bonded to the state that causes virus-specific immunologic tolerance in tolerance carrier induction people of virus antigen, and do not excite common immunoreation as defined above.Thereby the antigen of using by mucosa or Intradermal or upper intradermal routes, separately or combine with standard adjuvant, generally can excite common immunoreation, yet, its with as pharmaceutical composition of the present invention in causing and tolerate carrier and combine different.In these specific situations, in pharmaceutical composition of the present invention, cause tolerance carrier/antigen combined induction immunologic tolerance as defined above.This means that immunologic tolerance only can the suitable mixture that causes tolerance carrier and antigen (contain, or derived from Gag and/or the Pol of HIV virus) be realized by using (by mucosa or Intradermal or upper intradermal routes).If use a kind of and there is no another kind to people, people will be by " inoculation " or " by tolerating " so.
As shown in following embodiment (use SIV in model of rhesus monkey), by using according to immunologic tolerance (also referred to as " Ts " immunologic tolerance) pharmaceutical composition induction of the present invention or that realize, be characterised in that the CD8+T cell effect (particularly non-cell toxicity and/or MHC-1b/E restriction) that suppresses SIV Gag and/or Pol angtigen presentation CD4+T cell-stimulating (particularly activating in early days); And advantageously, be characterised in that one or more: the propagation that 1) there is no CD4+T cell; 2) under SIV antigenic stimulus, there is no the IFN-γ of significant CD8+T emiocytosis; With 3) the not remarkable anti-SIV IgM of whole body and the IgG antibody of producing.
By term " IFN-γ that there is no significant CD8+T emiocytosis under HIV or SIV antigenic stimulus ", the IFN-γ level that this means the CD8+T emiocytosis of observing in literary composition under HIV or SIV antigenic stimulus is 0 or weak.Under HIV or SIV antigenic stimulus, CD8+T emiocytosis IFN-γ is that " weak " is typically referred to as every 2 * 10
5individual PBMC is less than about 80SFC.
By term, " significantly do not produce the anti-HIV of whole body or anti-SIV IgM and IgG antibody ", in literary composition, mean that the anti-HIV of the whole body of observing or anti-SIV IgM and IgG antibody generation level are 0 or weak.It is that " weak " to be typically referred to as anti-HIV or anti-SIV titre be about 300 or still less that the anti-HIV of whole body or anti-SIV antibody produce.
Thereby, according to pharmaceutical composition induction of the present invention, preferably maintaining the antigen specific immune protection to HIV in people, wherein said immunoprotection preferred feature is:
-comparing with suitable experiment contrast, in described people, HIV virus load reduces, and preferably suppresses; And/or
-compare with suitable experiment contrast, in described people, suppress the CD8+T cell of HIV Gag and/or Pol angtigen presentation CD4+T cell-stimulating.
Advantageously, existence or activity by vitro detection from described people's CD8+ regulatory T cells, determine the described immunoprotection in described people.Can be by the ex vivo technique of standard, those that for example describe in following examples, carry out this detection.
All patents cited above, patent application and publication are incorporated to for referencial use in the text.
embodiment
the materials and methods that part A-is general
A-I animal.According to " Guide for the Care and Use of Laboratory Animals " regulations of state-run health research institute (National Institutes of Health), raise the common macaque of Chinese Rhesus Macacus (Macaca mulatta) of group support.All animal healths are good, and 2-4 year, body weight 4-6kg, is that SIV, SRV, monkey T cell are had a liking for lymphocyte virus 1, hepatitis virus B and B serum virus negative.When entering, all animals are carried out to X ray and tuerculoderma (PPD), to get rid of potential tuberculosis carrier.
A-II I class MHC typing.(people such as Muhl, 2002 as described above; The people such as Loffredo, 2007), use sequence specific primers (SSP) pcr analysis of representative Mamu-A and Mamu-B sequence carried out to gene type to Rhesus Macacus typical case I class MHC allele in peripheral blood lymphocytes (PBMC) sample.
The preparation of A-III antigenic virus.
III-1. with SIVmac239(P.A.Marx, presenting) inoculation CEM174 cell on carry out SIV production.Collect culture supernatant and select virus generation.
The SIVmac239:SIVmac239 of III-2.AT-2 inactivation is by 250 μ M aldrithiol(AT-) (Sigma) inactivation 2 hours, washs 3 times by ultracentrifugation.The virus of AT2 inactivation is used for using (that is, inoculation) with the final dose of 109 virions at every turn.
III-3. the SIVmac239:SIVmac239 of heat inactivation inactivation 30 minutes at 56 ℃.The virus of heat inactivation is with 10
9the final dose of virion is for use at every turn.
The SIVmac239:SIVmac239 of III-4.AT-2 heat inactivation is by 250 μ M aldrithiol(AT-2) (Sigma) inactivation 2 hours, washs 3 times by ultracentrifugation.Then, virus is accepted lower 30 minutes of 56 ℃ of temperature.The virus of inactivation is used for using with the final dose of 109 virions at every turn.
III-5. the virus formulation of inactivation is inoculated in to CEM174 cell, to confirm that 100% of viral infection suppresses.
The analysis of A-IV to the antibody response of SIV.By immunofluorescence antibody (IFA), analyze anti-SIV IgG, IgM and the IgA antibody in the titration blood plasma such as (Mederle people, 2003).In brief, at 37 ℃, the slide of the CEM174 cell adhesion that the test blood plasma of 2 times of serial dilutions and SIV infect is hatched 30 minutes.With after Hanks washing, the anti-macaque IgG(Sigma of goat that adds FITC-to put together), IgM(ADI, San Antonio, Texas) or IgA(ADI), then continue 30 minutes (in 37 ℃).Antibody titer is defined as reaching the inverse of the highly diluted of positive immunofluorescence dyeing.The sensitivity that IFA analyzes is 20 titres for IgG, for IgM and IgA, is 5 titres.When the IFA of plasma sample is feminine gender (below the sensitivity of analyzing), in order to contribute to data analysis, value is designated as 1.
People such as (, 1993) Tsai as described above, by splashing into conduit, rinse rectum with PBS and collect mucosa secretions with stomach.In brief, in sample, add trypsin inhibitor (10 μ g/ml) and EDTA(5 * 10-4M) (Sigma), then under 10000 * g, centrifugal 10 minutes at 4 ℃.Collect supernatant, and supplementary Phenylmethanesulfonyl fluoride (10-3M) and Hydrazoic acid,sodium salt (0.01%) are (Sigma).Sample is stored in to-80 ℃, until use.By the anti-SIV IgA titre in above-mentioned IFA analyzing and testing rectum.
A-V flow cytometry.With FACScalibur (BD Biosciences, San Jose, California), use anti-following fluorescently-labeled monoclonal antibody to carry out flow cytometry: CD3-PE-Cy7 (clone SP34-2), CD4-PE (clone MT477), CD8-PerCP (clone RPA-T8) and the anti-mice-APC bis-of rabbit anti-(BDBiosciences).The anti-P27 monoclonal antibody (Fitzgerald that Ki-67-PE (BD Biosciences) and FITC put together, Concord, MA) or coupling have the anti-P27 monoclonal antibody (Fitzgerald) of biotin-conjugated of APC-SAv (BD Biosciences) for the cell inner dyeing after saturatingization.
The monoclonal antibody that the PE of anti-TCR γ δ (clone B1), V β 8 and T cell differentiation antigen (CD7, CD16, CD28, CD62L, CD95, CD122, CD137, CD150, CD183, CD184, CD195, CD196, CD197, CD226, CD272 and CD305) puts together is purchased from BD Biosciences; The monoclonal antibody that the PE of anti-T cell differentiation antigen (CD11a, CD25, CD27, CD39, CD101, CD129, CD215, CD277 and CD357) puts together is purchased from BioLegend (San Diego, CA, USA); The monoclonal antibody of puting together with the PE of anti-T cell differentiation antigen (CD127, CD247 and CD279) is purchased from eBioscience (San Diego, CA, USA).
A-VI cell proliferation.People 2003 such as () Lu obtains PBMC as described above.By CF 5(6)-Carboxyfluorescein diacetate esters, succinimide ester (CFSE) labeled analysis (Molecular Probes, Eugene, Oregon), according to the description assessment SIV specific C D4+ of manufacturer or the propagation of CD8+T cell.With the 3 μ M CFSE PBMC15 minute that dyes at 37 ℃.After washing, with 10 μ g/ml restructuring SIV core protein P27 (ImmunoDiagnostics, Wobun, MA), 2 μ g/ml SIV gag15-mer peptides (GLS, Chinese Shanghai), 10
9the SIV of/ml AT-2 inactivation or independent medium were to the cytositimulation of CFSE labelling 5 days.With after anti-CD3 and anti-CD4 or anti-CD8 antibody labeling, fixing PBMC in 1% paraformaldehyde, for flow cytometry.
A-VII cell-stimulating.By magnetic bead, remove SIVmac239 that the fresh PBMC of (or not remove) CD8 or CD25 processed by AT-2 with 10
10the virus concentration single-wheel of/ml is infected 2 hours.With SEB (2.5 μ g/ml) and anti-CD3(2.5 μ g/ml)/anti-CD28(2.5 μ g/ml) the antibody cell that spends the night and stimulate to infect.Stimulate the rear cell inner dyeing that carries out SIV P27 and Ki-67 for 48 hours, to determine the interior percentage ratio that activates (Ki-67+) of (P27+) CD4+ cell of infection.
A-VIII ELISPOT analyzes.The test kit (Cell Sciences, Canton, MA) that uses business to sell when existing or not having the SIV of P27 or AT-2 inactivation, carries out Rhesus Macacus IFN-γ and IL-10ELISPOT and analyzes in the PBMC not cultivating.TGF-b1ELISPOT test kit is purchased from R & D Systems (Minneapolis, MN).With automatic ELISPOT reader (AID, GmbH, Stra β berg, Germany) reading out data.Non-specific SPC when deducting medium individualism, calculates the number of SIV specificity point formation cell (SFC).
A-IX antiviral is analyzed.When existing or not having the CD8+T cell of magnetic decontamination, with the ratio of the CD4/CD8 of 1:2, with SIVmac239 (10
-3mOI) the autologous homology CD4+T cell from each animal of actute infection magnetic positive mark (MicroBeads, Miltenyi Biotec) purification, then uses SEB(Sigma) stimulate 16 hours.After washing, in the RPMI1640 medium that contains 100IU people rIL2 (Invitrogen, Chinese Shanghai) of every Kongzui final volume 200 μ l, on 96 orifice plates, there is 5%CO
2time, at 37 ℃, quadruplicate cultured cell is 5 days.At the 3rd day, by the fresh medium of half, replace cell culture once.At the culture supernatant of collecting for the 5th day for measure virus load (as below) by real-time RT-PCR.By relatively from the geometrical mean that only contains virus concentration in the culture supernatant in duplicate hole of CD4+ infection cell, with the geometrical mean of virus concentration in the supernatant in quadruplicate hole from containing the CD8+ that mixes and CD4+ cell, calculate and suppress percentage ratio (%).In order to determine the dependency of virus between suppressing to limit with HLA, CD4+T cell also with allochthonous CD8+T co-culture of cells.
The measurement of A-X virus load.Use is to the primer of SIVmac239 and the special optimization of SIVmac251 (justice, SEQ ID No.1:5 '-GAGGAAAAGAAATTTGGAGCAGAA-3 '; Antisense, SEQ ID No.2:5 '-GCTTGATGGTCTCCCACACAA-3 ') and probe (SEQ ID No.3:5 '-FAM-AAAGTTGCACCCCCTATGACATTAATCAGATGTTA-TAMRA-3 '), by SIV RAN in real-time RT-PCR or the quantitative blood plasma of PCR or the relevant SIV DNA of cell.
The T cell analysis of the special inhibition of A-XI SIV.The scheme providing according to manufacturer (Miltenyi Biotec), the anti-CD8 puting together by magnetic bead or anti-CD 25 antibody are removed the fresh PBMC of (or not removing) CD8 or CD25, by SIVmac239, under 0.5 infection multiplicity (MOI), are infected 2 hours.With SEB (SEB) (2.5 μ g/ml) (Sigma) and anti-CD3(2.5 μ g/ml)/anti-CD28(2.5 μ g/ml) antibody (BD Biosciences) cell that spends the night and process to infect.Stimulate in vitro cell inner dyeing when carrying out SIV P27 and Ki-67 in latter 48 hours, to determine the percentage ratio (%) of the interior t cell activation (Ki-67+) of (P27+) cell mass of infection.
A-XII viral challenge.
XII-1. with SIVmac239(P.A.Marx, presenting) inoculation macaque PBMC on carry out SIV production.Collect culture supernatant and select virus generation.
XII-2. internal rectum challenge (IRC): after inoculation, animal internal rectum inoculation (repetition) 5000MID100, that is, and 5 * 10
5the pathogenic SIVmac239 of TCID50.The dosage of this infection causes the systemic infection of Chinese Rhesus Macacus 100% conventionally, between the 10th to 14 days, has plasma viral load peak value (10
6-10
7copy/ml).One month every two weeks, each month afterwards animal clinical and all SIV of ground assessment biology challenge.
XII-3. intravenous challenge (IVC): after inoculation, animal intravenous inoculation (repetition) 5MID100,, 500TCID50(titration in CEM174 cell line) pathogenic SIVmac239(is from Aaron Diamond AIDS Research Center, New York, doctor's P.A.Marx of USA present).The dosage infecting causes the systemic infection of Chinese Rhesus Macacus 100% conventionally, between the 10th to 14 days, has plasma viral load peak value (10
6-10
7copy/ml).One month every two weeks, each month afterwards animal clinical and all SIV of ground assessment biology challenge.
A-XIII statistical analysis.Before immunity and afterwards, respectively by azygous data or paired data between Mann-Whitney or the more different treated animals of Wilcoxon check.
part B-specific materials and method
B-I-BCG is as causing tolerance carrier
The preparation of B-I-I BCG
I-1. BCG lives: the BCG alive (SSI1331 strain) preparing at the Statens in Copenhagen Serum Institut is purchased from Laboratories Sanofi-Pasteur Merck, Sharp and Dome(is without translation in correspondence) (SPMSD), and with ultimate density 5 * 10
6cfu uses for enteral or intravaginal, or with ultimate density 5 * 10
5cfu strengthens using for each Intradermal.
I-2. expand the BCG of lyophilization (EFD) inactivation: the SSI133BCG strain of expansion lyophilization (EFD) killing living by 5 days under the vacuum that is less than 20 μ m Hg, with correspondence, each enteral or intravaginal uses 5 * 10
6cfu or each intradermal administration 5 * 10
5the final dose of cfu is used.
I-3. heat inactivation BCG: at 115 ℃, the SSI133BCG strain of living in the sterilizing of borate buffer solution mesohigh 15 minutes, each enteral or intravaginal uses 5 * 10 with correspondence
6cfu or each intradermal administration 5 * 10
5final dose use.
B-I-II pharmaceutical composition
Use contains SIV antigen and causes the fresh preparation compositions of RPMI1640 (Invitrogen Chinese Shanghai) of one of tolerance carrier.
B-I-III animal immune
When immunity, with tiletamine hydrochloride and zolazepam (0.7mg/kg) intramuscular injection anesthetized animal.
III-1. intravaginal immunity (IVI): inject 1 milliliter of pharmaceutical composition or above disclosed a kind of tolerance carrier that causes in contrast, immune jenny 4 hours by intravaginal under anesthesia.At the 8th week, in identical site, with identical dosage, give pharmaceutical composition or cause the booster immunization that tolerates carrier.After immunity for the first time, assess to every two weeks clinical and biologys all animals.
III-2. oral (gastric) immunity (IGI): take in pharmaceutical composition or a kind of as above disclosed in contrast cause tolerance carrier before, under anesthesia, to male or jenny gastric, use 15 minutes 15ml0.1M sodium bicarbonate.After using, give immediately other 15ml sodium bicarbonate solution.With the interval of month, to every animal, repeating twice inoculates with the first identical tolerance that causes.Latter every two weeks clinical and all animals of ground assessment biology of immunity for the first time.
III-3. Intradermal booster immunization (IDI): the 90th day after immunity for the first time, the Intradermal that gives animal (referring to above-mentioned IVI part) the 0.1ml pharmaceutical composition of female intravaginal immunity under anesthesia is strengthened, and described pharmaceutical composition contains 10
9the AT-2 inactivation SIV and 5 * 10 of copy
5the BCG alive of cfu.After immunity for the first time, assess to every two weeks clinical and biologys all animals.
B-I-IV antiviral is analyzed. and after corresponding internal rectum challenge, the threshold value of sterile immunity is at least 20.
b-II-Lactobacillus plantarum is as causing tolerance carrier
B-II-I antibacterial preparation (causing the preparation of tolerance carrier).Rotating speed with 200rpm at 37 ℃, on MRS medium is cultivated Lactobacillus plantarum (LP) (ATCC8014).For obtaining the LP of antibacterial culturing logarithm (in the middle of logarithm) phase, culture of bacteria is until the optical density under 600nm reaches 1.0, final LP concentration about 10
10cfu/ml(obtained at about 3.5 hours).
The animal immune that B-II-II is sent by oral (gastric).Animal overnight fast (there is no breakfast).When Orally administered, with tiletamine hydrochloride and zolazepam (0.7mg/kg) intramuscular injection anesthetized animal.
No.1:8 animal gastric of immunity used the preparation that 30ml virus-antibacterial is made, and it contains 4 * 10
7in copy/ml DI-SIV and maltodextrin (20%) solution 3 * 10
9the LP that cfu/ml lives.After this immunity for the first time, monkey is accepted virus-bacteria preparation that gastric 25ml is identical (that is, pharmaceutical composition) for every 30 minutes, continues 3 hours.This oral delivery scheme was carried out 5 times 5 continuous working days.In contrast, 4 animals are applied independent LP alive, only accept abreast inactivation SIV twice for other 3.
Immunity No.2:12 animal (iSIV/LP#9-20) gastric is used 30ml4 * 10
7copy/ml iSIV(AT-2/ heat inactivation SIVmac239) 3 * 10 in preparation and maltodextrin (20%) solution
9the LP that cfu/ml lives.Then, 5 continuous working days, animal per is accepted the preparation that 25ml is identical for 30 minutes, continues 3 hours (6 times).Six animals (LP#5-10) gastric is used 3 * 10 in 30ml maltodextrin (20%) solution
9the LP that cfu/ml lives.Then, 5 continuous working days, animal per is accepted the preparation that 25ml is identical for 30 minutes, continues 3 hours (6 times).Finally, other 6 animals (iSIV#5-10) gastric is used 30ml4 * 10
7the independent iSIV preparation of copy/ml.Then, 5 continuous working days, animal per is accepted the preparation that 25ml is identical for 30 minutes, continues 3 hours (6 times).
B-II-III removes CD8 in body
+t cell.First anaesthetize macaque, then, (the people such as Schmitz as described above, 1999), at the 0th, 4,7 days, give inosculating antibody CD8 monoclonal antibody (cMT-807, the Centocor Research & Development of intravenous injection 5mg/kg, Inc., Malvern, Pennsylvania, USA).Each time point after the injection of the 0th day and antibody is taked peripheral blood sample (5ml) from each animal.
B-II-IV antiviral is analyzed.Threshold value corresponding to the sterile immunity after internal rectum challenge is at least 100.
B-II-V CD8+ cell SIV inhibition analysis.There is or not exist the CD8 of magnetic purification
+during T cell, with the ratio of the CD4/CD8 of 1:3, with SIVmac239 (10
-3infection multiplicity) the autologous homology CD4 from each animal of actute infection magnetic positive mark (MicroBeads, Miltenyi Biotec) purification
+t cell, then stimulates 16 hours with SEB and anti-CD3/ anti-CD28 antibody.After washing, quadruplicate cultured cell on 96 orifice plates.In the RPMI1640 medium that contains 100IU people rIL2 (Roche Diagnostics GmbH, Mannheim, Germany) of every Kongzui final volume 200 μ l, culture maintains 5 days.The culture supernatant of collecting at the 5th day is for measuring virus load (seeing following) by real-time RT-PCR.As following calculating suppresses multiple: from only infecting the geometrical mean of virus concentration in the culture supernatant of CD4+ target cell/from the geometrical mean of virus concentration in the CD8+ mixing and CD4+T cell conditioned medium).
In some experiments, by using Multiwell Insert System (BD Biosciences) there is no cell-cells contacting, cultivate CD8+ and CD4+T cell (CD8 is at patchhole, and CD4 is in the hole of bottom); CD4+T cell and allochthonous CD8+T co-culture of cells, to determine the dependency between virus inhibition and MHC restriction; CD8+ and CD4+T cell are also cultivated altogether when anti-MHC-ABC (BioLegend) or anti-MHC-E (Cell Science) antibody exist, to define the pattern of MHC restriction.In order to define the CD8+T cell subsets (subset) relevant to antiviral activity, use by the anti-PE microballon of LD post (Miltenyi Biotec), after puting together anti-TCR γ δ, anti-V β 8 or other anti-T cell differentiation antigen antibody and remove with PE, immediately from PBMC purification CD8+T cell.
The cytotoxicity analysis of B-II-VI SIV specific C D8+T cell.The CD8+T cell (effector lymphocyte) of purification and with 10
2the CD4+T cell (target cell) of the purification of the SIVmac239 pulse that AT-2 processes is used 40nM3,3 ' bis-hexyloxy carbocyanine (DiOC at 37 ℃
6) (people such as Marchetti, 1996) (Molecular Probes) labelling 10 minutes.The anti-CD4 that target cell is puted together with PerCP-Cy5 on ice (BD Bioscience) labelling 20 minutes.Wash after 3 times, in 96 orifice plates at the bottom of U with different E/T than (3:1,1:1,0.3:1) melange effect cell and target cell in triplicate.From 4 healthy donors, there is anti-CD32 (BD Bioscience) that APC puts together and the CD56 of purification
+(NK) cell (effector) is included into the contrast performing an analysis.When SEB and the anti-CD28 of anti-CD3/ exist, at 37 ℃, hatch after 4 hours, harvesting, and by flow cytometry.The following cytotoxicity percentage ratio that calculates: 100 * (target cell of the total apoptosis target cell-% of % spontaneous apoptosis)/(target cell of 100-% spontaneous apoptosis).
The challenge of B-II-VII virus.
Research first: in first experiment (immune No.1), Orally administered vaccine or contrast latter 4 months, 8 immune animals and its 7 contrast internal rectum inoculation 2500MID
100(100.000TCID
50) pathogenic SIVmac239.After two months, 4 inoculations and protected monkey by internal rectum approach, again challenge (100.000TCID
50), and other 4 protected monkey 5MID
100(200TCID
50) SIVmac239 intravenous challenges again.In contrast, 2 monkeys are accepted internal rectum challenge, accept intravenous challenge for other 2.The dosage of these infection caused the systemic infection of 100% Chinese Rhesus Macacus conventionally between the 10th to 14 days, and plasma viral load reaches peak value (10
7-10
9vp/ml).
Research (immune No.2) for the second time: in second group of research after immunity the 420th day, 16 animals (with 8 monkeys of iSIV and LP immunity) and 100,000TCID for 8 contrasts (4 iSIV and 4 LP)
50sIVmac239 internal rectum challenge.
part C-result
c-I BCG causes tolerance carrier
the rear protection to intravenous SIVmac239 challenge is used in C-I-I intravaginal:
Six animals (compositions _ 1,2,3,4,5 and 6) intravaginal is used 1 milliliter and is contained and cause tolerance compositions as the virus of the AT2 inactivation of antigen and the BCG that lives.At the 8th week, at same loci, by the identical tolerance compositions that causes, strengthen using.
Side by side, 5 other animal (contrast _ 1,2,3,4 and 5) intravaginal give 1 milliliter only containing the compositions of the BCG that lives.Also at the 8th week, in identical site, by identical compositions, strengthen using.
Use for the first time latter 4 months, all 11 animals (compositions _ 1-6 and contrast _ 1-5) are challenged by intravenous virus inoculation.
In the blood plasma of processing animal, measure virus load routinely.
Fig. 1 shows in the animal of accepting compositions (compositions _ 1-6) and in control animal (contrast _ 1-5), after the challenge of single dose intravenous inner virus, virus load (blood plasma SIV RNA copy/ml) as the time (my god) function.
Result shows, after intravenous viral challenge, 5 control animals (contrast _ 1-5) show typical primary infection, as expected, have plasma viral load peak value (10 after challenge between 10-14 days
6-10
7copy/ml).The plasma viral load of this control animal group after viral challenge 60 days and still continue thereafter high (>10
5vp/ml).
On the contrary, 4/6 the animal that intravaginal is accepted to be made by the SIVmac239 of AT-2 inactivation and BCG causes tolerance compositions shows low-down plasma viral load peak value (between 10-14 days, <1000vp/ml), it becomes very soon and can not detect (<10vp/ml) (after viral challenge 1 month).At the 60th day, there is high plasma viral load peak value (>10
6copy/ml) 2 animals are than matched group (>10
5copy/ml) there is lower fixed point (set-point) virus load level (<1000 copy/ml).
the protection of after compositions, internal rectum SIVmac239 being challenged is used in C-I-II-intravaginal:
Seven animals (compositions _ 7,8,9,10,11,12 and 13) intravaginal is used 1 milliliter and is contained as the virus of the AT2 inactivation of antigen with as the compositions that causes the BCG alive of tolerance carrier.At the 8th week, in identical site, by identical compositions, strengthen using.
Side by side, 5 other animal (contrast _ 6,7,8,9 and 10) intravaginal give 1 milliliter only containing the compositions of the BCG that lives.Also in the 8th week identical site, by identical compositions, strengthen using.
Use for the first time latter 4 months, all 12 animals (compositions _ 7-13 and contrast _ 6-10) are challenged by SIVmac239 by internal rectum virus inoculation.
In that process and blood plasma control animal, measure routinely virus load.
Fig. 2 shows after internal rectum viral challenge, in the animal of accepting compositions (compositions _ 7-13) and in control animal (contrast _ 6-10), virus load (blood plasma SIV RNA copy/ml) as the time (my god) function.
Result shows, after internal rectum viral challenge, accepts 5 animals (contrast _ 6-10) that independent BCG intravaginal alive uses and shows typical primary infection, as expected, has plasma viral load peak value (10 after challenge between 10-14 days
6-10
7vp/ml).The blood plasma fixed point virus load of this control animal group still continues high (>10 for 60 days after viral challenge
5copy/ml).
On the contrary, 4/7 the animal that the SIVmac239 of AT-2 inactivation and the BCG that lives are accepted in intravaginal shows the virus load level that can't detect (< 10 copies/ml) during after challenge 60 days astoundingly.3 other animals show typical primary infection, have plasma viral load peak value after challenge between 10-14 days.But, its virus load (10 of fixing a point
3-10
5copy/ml) the remarkable level (>10 lower than control animal
5).
the protection of challenging repeating intravenous or internal rectum SIVmac239 after C-I-III-intravaginal drug administration compositions:
Two and after eight months, after intravenous challenge, have the virus load that can not detect 3 animals (compositions _ 1, _ 2 and _ 3) accept second and intravenous challenge for the third time of same dose virus inoculation thing.
This group monkey second and for the third time after intravenous viral challenge, at the 10th day, observe similar low plasma viral load peak value.But by after viral challenge 30 days, virus load becomes again can not detect (Fig. 3).
After compositions initial application 16 and 23 months, had altogether 3 intravenous challenges 3 animals (compositions _ 1, _ 2 and _ 3) again by internal rectum inoculation challenge.
As expected, after 2 continuous internal rectum viral challenges, these 3 animals (it is initially accepted intravaginal AT-2-inactivation SIVmac239 and adds BCG) show (<10 copy/ml) plasma viral load (Fig. 3) can't detect again.
These results are established, and the efficiency that suppresses virus replication is stable, and reason is that this is suppressed at initial composition and uses and still can observe above for latter 20 months.
after C-I-IV-intravaginal drug administration compositions and Intradermal are strengthened, the protection to intravenous or internal rectum SIVmac239 challenge:
As expected, follow intravenous (contrast 17 and 18, Fig. 4) or internal rectum (contrast 19 and 20, Fig. 5) after viral challenge, accept the animal that work BCG intravaginal is used separately for 4 and show typical primary infection, as expected, after challenge, between 10-14 days, there is plasma viral load peak value (10
6-10
7vp/ml).The blood plasma fixed point virus load of this control animal group is to 60 days high (>10 still after viral challenge
5copy/ml).
On the contrary, intravaginal accept the compositions made by the SIVmac239 of AT-2 inactivation and the BCG that lives and with the 3/4(75% of same combination Intradermal reinforcement) animal ( compositions 14,15 and 17), during intravenous is challenged latter 60 days, the plasma viral load (<10 copy/ml) (referring to Fig. 4) that performance can't detect.Remaining 1 animal (compositions 16) performance primary infection has plasma viral load peak value (>10 after challenge between 10-14 days
5copy/ml) (referring to Fig. 4).But its fixed point virus load reached relatively low level (10 at the 60th day
4copy/ml).
And, accept compositions that intravaginal made by the SIVmac239 of AT-2 inactivation and the BCG that lives and with the 4/4(100% of same combination Intradermal reinforcement) animal (compositions 18-21), during internal rectum is challenged latter 60 days, the plasma viral load (<10 copy/ml) (referring to Fig. 5) that performance can't detect.
protection to internal rectum SIVmac239 challenge after the Orally administered pharmaceutical composition of C-I-V-:
4 animals (compositions _ 22, _ 23, _ 24 and _ 25) gastric uses 1 milliliter of virus that contains AT2 inactivation and the compositions of the BCG that lives.
Side by side, 4 other animal (contrast 21-24) gastric give 1 milliliter of independent BCG alive.
Give for the first time identical the using of each animal, after step of applying for the first time the 15th day, 30 days and 60 days, then repeat 3 times.
Result shows, after internal rectum viral challenge (carrying out at the 90th day), 4 animals (contrasting 21-24) of accepting independent BCG alives show typical primary infection, have plasma viral load peak value (10 after challenge between 10-14 days
6-10
7and the animals (compositions _ 22-25) of the compositions of 4 SIV that accept AT-2 inactivation and the BCG that lives show the plasma viral load (<10 copy/ml can't detect astoundingly copy/ml); Between 10-14 days) (Fig. 6).
c-I-VI-uses Immune interrelation and the protection to SIVmac239 challenge after the compositions of being made by the virus of AT2 inactivation and the BCG that lives:
In the blood of processing animal, the whole body antibody for SIV do not detected.But, when using Intradermal reinforcement compositions to use, some specificity whole body humoral responses detected.Thereby what observe is not the humoral response from whole body to processing the protection of the anti-SIV infection of animal.
And, the cytotoxic T lymphocyte (data do not show) of conventional generation SIV specificity IFN-γ by ELIspot, do not detected.The object whether existing in order to assess the unconventional cell effect of SIV specificity, extracts blood sample to each processing or control animal, according to conventional methods from each Sample Purification on Single CD4+ and CD8+ cell.Cultivate the CD4+ cell obtaining in advance, then with SIV mac239, infect according to conventional methods.Then, when there is or not existing the autologous homology CD8+ cell of prior acquisition, cultivate the CD4+ cell 5 days that SIV infects.By quantitative PCR in real time, analyze supernatant SIV concentration.
During autologous homology CD8+ cell that Fig. 7 shows to obtain in the process of the experiment that exists or do not exist Fig. 2 to represent, the inhibition multiple of virus replication in the CD4+ that the SIV of acquisition infects.The CD8 of test is available from accepting the virus of AT2 inactivation and the animal of BCG compositions (compositions _ 7-13) by intravaginal approach or from control animal (contrast 6-10).
In the cd4 cell that result shows to infect at SIV from the protected cd8 t cell of avoiding the animal (compositions _ 7, compositions _ 8, compositions _ 10 and compositions _ 11) of viral infection, provide the virus inhibition level that is greater than 20 times, and from not having the protected cd8 t cell of avoiding the animal (compositions _ 9, compositions _ 12 and compositions _ 13) of viral infection that the virus inhibition level (Fig. 7) that is less than or equal to 10 times is provided.And, also in 4 animals of intravenous viral challenge are avoided in protected shown in Fig. 1, observe and be greater than the virus of 20 times and suppress (data do not show).
During autologous homology CD8+ cell that Fig. 8 shows to obtain in the process of the experiment that exists or do not exist Fig. 6 to represent, viral inhibition level in the CD4+ that the SIV of acquisition infects.The CD8 of test obtains from the virus by Orally administered AT2 inactivation and BCG and accepts 4 animals of compositions (compositions _ 22-25) or from 4 control animals (contrast 21-24).
The SIV(P27+ obtaining during autologous homology CD8+ cell that Fig. 9 shows to obtain in the process of the experiment that exists or do not exist Fig. 6 to represent) t cell activation (Ki-67+) level in the cd4 cell group who infects.The CD8 of test obtains from the virus by Orally administered AT2 inactivation and BCG and accepts 4 animals of compositions (compositions _ 22-25) or from 4 control animals (contrast 21-24).The SIV specificity of observing in 4 animals of accepting compositions by the CD4+T cell-stimulating of autologous homology CD8+T cell suppresses.
These results verifications, per vaginam interior or Orally administered AT2 inactivation and the whole body of BCG acquisition or the prevention that mucosa SIV infects, induced a kind of immune tolerance state, this immune tolerance state is characterised in that the CD8+T cell effect of the non-cell toxicity relevant to the cd4 cell anergy of SIV infection.In conjunction with these results, thereby BCG is accredited as and causes tolerance adjuvant.
In a word, these find to have confirmed for the first time that by intravaginal or oral (or gastric), using the compositions of being made by the SIV virus of inactivation and the BCG that lives realizes the steady statue to the tolerance of SIV antigen immune.Meanwhile, its pharmaceutical composition that also shows for the first time intravaginal or the Orally administered SIV according to the AT2 of containing inactivation of the present invention virus and the BCG that lives chronic viral infection after (>50%) prevention internal rectum or intravenous challenge effectively.
c-II Lactobacillus plantarum is as causing tolerance carrier
c-II-I-uses the SIV of two inactivations and the SIV specific immunologic tolerance of Lactobacillus plantarum (iSIV/VP) induction by oral combination
On the one hand, (Figure 10 a) in the animal of processing with oral iSIV/LP, SIV specific antibody (IgG, IgM and IgA) not detected.On the other hand, in the animal of processing at iSIV/LP, do not observe significant SIV P27-specificity peripheral blood CD4+T cell proliferation, and the animal that iSIV processes shows significant P27 specificity peripheral blood CD4+T cell proliferation.
anti-activation and the antiviral activity of C-II-II-non-cell toxicity CD8+ cell
In the animal of processing with iSIV of processing at iSIV/LP, all observe SIV P27-specificity peripheral blood CD8+T cell proliferation (Figure 10 b).But, IFN-γ secretion T cell (at extracellular stimulus time) in the animal that iSIV/LP processes, do not detected, remove the anergy (Figure 10 c) that CD8+ or CD25+ cell can not change P27-specific effector T cells.And, in the PBMC of acute Infection in Vitro that processes animal from iSIV/LP, also observe by non-cell toxicity CD8+T cell and suppress consumingly (P27+) CD4+T cell-stimulating (Ki-67+) infecting, remove CD25+ cell and do not change the strong inhibition (Figure 10 d) to the CD4+T cell-stimulating infecting that CD25-CD8+T cell produces.
Note such fact: when existing or not having SEB and anti-CD3/ anti-CD28 antibody, after jointly hatching CD8+T cell and the CD4+T cell with the non-SIVmac239 pulse copying, by highly sensitive cytotoxicity analysis (people such as Marchetti, 1996), lysis (Figure 10 e) do not detected.
Finally, the peripheral blood CD8+T cell of processing the animal of >=2 months from iSIV/LP shows the inhibition of virus replication in the autologous homology CD4+T cell of the strong acute Infection in Vitro of antagonism, and active (Figure 11 a).And, in the allos CD4+T of acute Infection in Vitro cell, also observe the antiviral activity that this CD8+T cell is strong (Figure 11 b), show that non-classical HLA1 restriction scheme participates in the inhibition of CD8+T cell/inhibition active.
Take from the purification peripheral blood CD8+T cell with the macaque of a LP/iSIV >=2 month Initial stage of immunization, the CD4+T cell that the acute SIVmac239 of autologous homology is infected (with the stimulation of spending the night of SEB and anti-CD3/ anti-CD28 antibody, and co-cultivation 5 days) has strong antiviral activity.Once SIV specific C D4+T cell-stimulating is set up (stimulating latter 48 hours), add CD8+T cell and no longer can suppress virus replication (Figure 11 c).People such as (, 2010) Jiang showing in the research of people's 1 type autoimmune type 2 diabetes mellitus as in the previous made excuses in this observation, and in the cultivation extending, CD8+T cell is to target (effectively (productively) infects) the potential cracking of CD4+T cell.The cell-mediated antiviral activity of this CD8+T needs cell-intercellular contact (Figure 11 d), and be that classical MHC1a is unrestriced, (Figure 11 e) as shown in the inhibition of the strong virus replication by from being produced by CD8+T cell on other immune animal or the CD4+T cell from the actute infection of control animal.Finally, the antiviral activity of CD8 mediation is by anti-MHC-1b/E antibody blocking, and not by anti-MHC-1a/ABC antibody blocking, shows the CD8+T cytoactive (Figure 11 f) of non-classical MHC-1b/E restriction.
Having established CD8+T cell TCR, to express the MHC-1b/E peptide complexes that identification is carried by target CD4+T cell be necessary (people such as Sarantopoulos, 2004; Van Kaer, 2010).Use is removed outward by puting together the magnetic microsphere of antibody, shows that TCR γ δ and V β 8 do not participate in the CD8+T cell inhibition (Figure 11 g) of virus replication.Thereby, in the MHC-1b/E peptide of offering on the CD4+T cell that TCR α β seems to infect in identification, play an important role.And, by using with non-human primates film CD(referring to " differentiation group (Cluster Differentiation) ") (CD7, CD11a, CD16, CD25 (IL-2RA), CD27, CD28, CD39, CD62L, CD95, CD101, CD122 (IL-2RB), CD127 (IL-7R), CD129 (IL-9R), CD137, CD150, CD183 (CXCR3), CD184 (CXCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CD215 (IL-15Ra), CD218 (IL-18Ra), CD223 (LAG3), CD226, CD247, CD272, CD277, CD279 (PD-1), the anti-human antibody obtaining of CD305 (LAIR1) and CD357) antigenic cross-reaction is removed CD8+T cell, do not identify the T cell differentiation antigen (table 1) relevant to the CD8+T cytoactive of MHC-1b/E restriction.* before the subgroup that following table 1 shows in first immunisation research (immune No.1), limits at T cell differentiation antigen is removed and afterwards, takes from the antiviral activity (suppressing multiple, geometrical mean ± SE) of the CD8+T cell of 8 iSIV/LP immune animals.
Table 1
* at each batch of experiment, carry out from antiviral activity (inhibition multiple, geometrical mean ± SE 8 iSIV/LP immune animals, remove the CD8+T cell of (or not removing) with the antibody of 2 anti-T cell differentiation antigens.
c-II-III is watched for animals and is avoided internal rectum challenge by oral tolerance
Use after oral iSIV/LP or control formulation 3 months, with single high dose (100,000TCID
50) 8 iSIV/LP immunity of SIVmac239 internal rectum challenge with 7 control animals.8 iSIV/LP process 8 protected internal rectum challenges that avoid pathogenic SIVmac239 in animal, and the animals with 4 LP processing of 4 iSIV processing are infected (Figure 12 a and b, the left half of figure) by identical internal rectum viral challenge.
c-II-IV is watched for animals and is avoided intravenous challenge by oral tolerance
Challenge for the first time latter two month, by intravenous route, accept to challenge for the second time (200TCID for 4 in 8 monkeys
50).After challenge the 10th day, they are all showed the slight peak value (≤200SIV DNA copy/hundred ten thousand PBMC and 200SIV RNA copy/ml blood plasma) that copies; But by the 30th day, PBMC SIV DNA was reduced to≤10 copy/hundred ten thousand cells, and blood plasma SIV RNA can not detect (≤10 copies/ml), shows not have the active replication (Figure 12 a and b, the right half of figure) of body inner virus.On the contrary, accept identical intravenous SIVmac239 challenge (200TCID
50) 2 untreated animals successfully infected.4 remaining monkeys are challenged (100.000TCID again by internal rectum
50), it is all by protection (Figure 12 a and b, the right half of figure) completely.
c-II-V confirms the effect of CD8+T cell in body
This is challenged latter 5 months for the second time, in order to confirm to act in the body of CD8+T cell, during one week, (immune latter the 300th, 304 and 307 days) give the anti-CD8 antibody of mice-people chimeric monoclonal (cMT-807 3 times to 8 monkeys of having been challenged, Centocor) intravenous injection, temporarily to remove its CD8+T cell (Figure 13 a & b) from its peripheral blood and lymphoid organ.The appearance of viral RNA or DNA in 4 macaques that rechallenged by internal rectum approach, do not detected, prove again its sterility protection completely; On the contrary, if its plasma viral load peak value is 10
6rNA copy/ml, with and PBMC and lymph node provirus carrying capacity to the within 15 days, reach 10
4dNA copy/10
6shown in individual cell (minimum point that CD8+T cell is removed), from the animal lymph sample organ of 4 intravenous challenges, remove CD8+T cell and follow strong virus replication; To 60-90 days, when 4 monkeys recover baseline CD8+T cell concentration, blood plasma SIV RNA and PBMC and lymph node SIVDNA also return to baseline values (Figure 13 c, d & e).This CD8+T cell of having confirmed iSIV/LP induction, controlling the unique effect of intravenous SIV challenge internal animal virus in copying, is wherein inferred and is had the virus lays dormant of replication capacity in static memory CD4+T cell.
Challenge for the second time latter 8 months, 4 monkey and 4 monkey acceptance challenges for the third time that intravenous rechallenges that internal rectum rechallenges, this time with SIVB670(100,000TCID
50) a kind of different infectious SIV strain, per rectum approach.As its SIVB670DNA can't detect and rna level; and 2 untreated animals are challenged successfully shown in infection by identical SIVB670; 12 months 8 animals have subsequently retained protection completely; this shows the CD8+T cell of the MHC-1b/E restriction that LP/iSIVmac239 produces; by the CD4+T cell-stimulating that prevents from being infected by other SIV strain, be (Figure 14 a & b) of cross protection.
In order to determine the valid period of preventing SIV disease in the animal that iSIV/LP processes, in the macaque in 8 new China sources, carried out the immunity for the second time with iSIV/LP, do not have SIV challenge to detect the extracorporeal antivirus effect of its CD8+T cell in time active.With with LP(n=4) or independent iSIV(n=4) the control animal comparison processed, detected from this extracorporeal antivirus effect of latter 60 days of immunity active.
In 8 monkeys 7 until kept in vitro anti-SIV activity level on the 420th day, and the antiviral activity of 1 monkey declined progressively from the 360th day, and (Figure 15 a) by the 420th day, to reach the baseline values of contrast monkey.After immunity the 420th day, with 100,000TCID
5016 animals of SIVmac239 internal rectum challenge.7 in 8 iSIV/LP immune animals have obtained sterile immunity, in blood plasma and PBMC (Figure 15 b & c) and in rectal mucosal lymphocyte in (its from challenging the 1st day measure) and pelvic lymph node (Figure 16 a to 16e) without any SIV RNA and DNA appearance, and 1 immune monkey infects completely.Importantly, the evolution of the in vitro antiviral activity of 8 inoculation monkeys allows within latter 360 days, to predict (that is, it is challenged first 60 days) (Figure 15 a to c) according to 7 protected monkeys and 1 not protected monkey immunity.
c-II-VI conclusion
In literary composition, disclose; in model of rhesus monkey; the SIVmac239(iSIV of inactivation) and aulophyte lactobacillus (LP) (be called cause tolerance adjuvant) use the CD8+T cell that has produced MHC-1b/E restriction; the inhibition of its induction SIV angtigen presentation CD4+T cell-stimulating; thereby suppress SIV, copy, and protect macaque to avoid SIV challenge.
The mixture gastric of being made by iSIV and the LP of inactivation is applied to 16 animals and 15 contrasts altogether.After 4 to 14 months, with pathogenic SIVmac239 internal rectum, challenge all animals.
In the animal that 16 iSIV/LP use, 15 merely hit and observe anti-SIV completely and infect protection; On the contrary, in all control animals and 1 inoculation monkey, established infection.At internal rectum, challenging first 60 days can be by the not protected monkey of in vitro antiviral analyses and prediction.In 4 monkey medium-sized veins, give internal rectum in SIVmac239 challenge for the second time and other 4 monkeys and give after SIVmac239 challenge for the second time, 8 protected animals have retained protected property.
The monkey that 8 iSIV/LP send does not have SIV specificity peripheral blood CD4+T cell proliferation completely, and does not produce the SIV specific antibody (IgG, IgM or IgA) of any whole body.
And its SIV specificity peripheral blood CD8+T cell has Some features:
1) but while stimulating in vitro, it is bred well and be there is no IFN-γ secretion;
2) it suppresses the activation of the autologous homology CD4+T cell of actute infection consumingly;
3) removing CD25+ cell latter two function all remains unchanged;
4) it also suppresses SIV in the allogeneic CD4+T cell of actute infection and copies; With
5) its inhibition/inhibitory action is MHC-1b/E restriction.
These results show that the co-administered iSIV of gastric and LP allow macaque to produce the CD8+ regulatory T cells of virus-specific non-cell toxicity MHC-1b/E restriction; it produces SIV specific immunologic tolerance; and very astoundingly, this virus-specific immunologic tolerance is relevant to the vaccine protection that anti-SIV infects the animal of establishing.
More than show according to the infection of HIV and SIV in pharmaceutical composition prevention people/mammal of the present invention.The effect of this prevention causes " Ts " immunologic tolerance in the experimenter's (that is, having used the mammal of pharmaceutical composition) who tolerates inoculation and obtains in macaque by induction.Described " Ts " immunologic tolerance literary composition middle finger comprises the inhibition CD8 regulatory T cells of virus-specific non-cell toxicity MHC-1b/E restriction, and it exists and activity shows:
-suppress to have used copy (external) of SIV in the CD4+T cell of actute infection of macaque of pharmaceutical composition of the present invention; And/or
-prevention copies (in body) with SIV in the macaque that causes tolerance inoculation of the SIV challenge of infecting.
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Claims (33)
1. a pharmaceutical composition, the mixture that it comprises particulate antigen and non-pathogenic bacteria.
2. pharmaceutical composition according to claim 1, wherein said antigen has one or more epi-positions from HIV Gag and/or Pol albumen.
3. a pharmaceutical composition, its comprise have one or more from HIV Gag and/or the antigen of Pol albumen epi-position and the mixture of non-pathogenic bacteria.
4. pharmaceutical composition according to claim 3, wherein said antigen is particulate antigen.
5. according to the pharmaceutical composition described in claim 1 to 4 any one, wherein said antigen is 110kDa size at least approximately.
6. according to the pharmaceutical composition described in claim 1 to 5 any one, wherein said particulate antigen is selected from virion, virus-like particle, recombinant virus particle, the virus protein of puting together and concatemer virus protein.
7. pharmaceutical composition according to claim 6, wherein said recombinant virus particle or described virion are inactivations.
8. according to the pharmaceutical composition described in claim 2 to 7 any one, the wherein said epi-position from HIV Gag albumen is selected from from the epi-position of capsid protein (p24) with from the epi-position of stromatin (p17).
9. according to the pharmaceutical composition described in claim 2 to 7 any one, the wherein said epi-position from HIV Pol albumen is selected from epi-position from intergrase, from the epi-position of reverse transcriptase with from the epi-position of protease.
10. according to the pharmaceutical composition described in claim 1 to 9 any one, wherein said antibacterial is alive.
11. according to the pharmaceutical composition described in claim 1 to 10 any one, and wherein said antibacterial is selected from the malignant bacteria of attenuation, (optionally, being also attenuation) malignant bacteria and the non-pathogenic lactobacillus of inactivation.
12. pharmaceutical compositions according to claim 11, wherein said antibacterial is lactobacillus.
13. pharmaceutical compositions according to claim 12, wherein said antibacterial is Lactobacillus plantarum.
14. according to the pharmaceutical composition described in claim 6 to 13 any one, and wherein said particulate antigen is virion, and wherein the amount of virion is mixture about 10 described in every ml
6to about 10
12.
15. according to the pharmaceutical composition described in claim 1 to 14 any one, and the amount of wherein said antibacterial is mixture about 10 described in every ml
4to about 10
14cFU.
16. according to the pharmaceutical composition described in claim 6 to 15 any one, and wherein said particulate antigen is virion, and virion described in wherein said mixture is that about 1:10 is to about 1:1000, preferably approximately 1:100 to the ratio of described antibacterial.
17. according to the pharmaceutical composition described in claim 1 to 16 any one, and it is mucosa or Intradermal or intraepithelial pharmaceutical composition.
18. pharmaceutical compositions according to claim 17, it is combination of oral medication.
19. according to the pharmaceutical composition described in claim 1 to 18 any one, and it is for preventing and/or treating in the method for the people HIV disease needing.
20. according to the pharmaceutical composition described in claim 1 to 18 any one, and it is avoided in the method for HIV for the protection of people.
21. according to the pharmaceutical composition described in claim 1 to 18 any one, and it is avoided in the method for HIV seroconversion for the protection of people.
22. according to claim 19 to the pharmaceutical composition described in 21 any one, and wherein said particulate antigen is virion, wherein to described people, uses about 10 every day
8to about 10
14the dosage of individual virion.
23. according to claim 19 to the pharmaceutical composition described in 22 any one, wherein use about 10 every day to described people
6to about 10
16the dosage of antibacterial described in CFU.
24. according to the pharmaceutical composition described in claim 1 to 23 any one, and it induces and preferably maintain the antigen specific immune protection that resists HIV in people.
25. pharmaceutical compositions according to claim 24, wherein said immunoprotection is characterised in that and reduces and preferably suppress the HIV virus load in described people.
26. according to the pharmaceutical composition described in claim 24 or 25, and wherein said immunoprotection is characterised in that the activation that is suppressed HIV Gag in described people and/or Pol angtigen presentation CD4+T cell by CD8+ regulatory T cells.
27. pharmaceutical compositions according to claim 26 are wherein determined described immunoprotection by vitro detection from the existence of described people's CD8+ regulatory T cells or activity in described people.
28. according to the pharmaceutical composition described in claim 26 or 27, and wherein said CD8+ regulatory T cells is the CD8+ regulatory T cells of MHC-1b/E restriction.
29. according to the pharmaceutical composition described in claim 2 to 28 any one, wherein said HIV is HIV-1 or HIV-2.
30. 1 kinds for preventing and/or treating the pharmaceutical kit of the people HIV disease needing, and it comprises:
-particulate antigen in the first container; With
-non-pathogenic antibacterial alive in second container,
Described antigen and described antibacterial for mucosa or Intradermal or on the pharmaceutically acceptable carrier of intradermal administration.
31. pharmaceutical kit according to claim 30, wherein said antigen has one or more epi-positions from HIV Gag and/or Pol albumen.
32. 1 kinds for preventing and/or treating the pharmaceutical kit of the people HIV disease needing, and it comprises:
-antigen in the first container, described antigen has one or more epi-positions from HIV Gag and/or Pol albumen, and
-non-pathogenic antibacterial alive in second container,
Described antigen and described antibacterial for mucosa or Intradermal or on the pharmaceutically acceptable carrier of intradermal administration.
33. pharmaceutical kits according to claim 32, wherein said antigen is particulate antigen.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510856888.3A CN105709221B (en) | 2011-04-06 | 2012-04-06 | Pharmaceutical composition for the prevention and/or treatment of HIV diseases in humans |
| CN201711347221.6A CN107899009B (en) | 2011-04-06 | 2012-04-06 | Pharmaceutical composition for the prevention and/or treatment of HIV diseases in humans |
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| CNPCT/CN2011/072481 | 2011-04-06 | ||
| CNPCT/CN2011/072481 | 2011-04-06 | ||
| US201161534088P | 2011-09-13 | 2011-09-13 | |
| US61/534,088 | 2011-09-13 | ||
| CNPCT/CN2012/070761 | 2012-01-30 | ||
| CNPCT/CN2012/070761 | 2012-01-30 | ||
| US201261609051P | 2012-03-09 | 2012-03-09 | |
| US61/609,051 | 2012-03-09 | ||
| PCT/IB2012/000857 WO2012137071A2 (en) | 2011-04-06 | 2012-04-06 | Pharmaceutical compositions for preventing and/or treating an hiv disease in humans |
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| CN201510856888.3A Division CN105709221B (en) | 2011-04-06 | 2012-04-06 | Pharmaceutical composition for the prevention and/or treatment of HIV diseases in humans |
| CN201711347221.6A Division CN107899009B (en) | 2011-04-06 | 2012-04-06 | Pharmaceutical composition for the prevention and/or treatment of HIV diseases in humans |
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| CN201280026590.4A Expired - Fee Related CN103732248B (en) | 2011-04-06 | 2012-04-06 | Pharmaceutical composition for preventing and/or treating the HIV diseases in people |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5895758A (en) * | 1997-06-10 | 1999-04-20 | Bio-Energy Systems | Strain of lactobacillus Plantarum |
| CN1387442A (en) * | 1999-09-17 | 2002-12-25 | 荷兰应用自然科学研究组织 | Oral recombinant lactobacilli vaccines |
| CN101291691A (en) * | 2003-08-28 | 2008-10-22 | 免疫反应公司 | Immunogenic HIV compositions and related methods |
-
2012
- 2012-04-06 CN CN201711347221.6A patent/CN107899009B/en not_active Expired - Fee Related
- 2012-04-06 CN CN201280026590.4A patent/CN103732248B/en not_active Expired - Fee Related
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2013
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5895758A (en) * | 1997-06-10 | 1999-04-20 | Bio-Energy Systems | Strain of lactobacillus Plantarum |
| CN1387442A (en) * | 1999-09-17 | 2002-12-25 | 荷兰应用自然科学研究组织 | Oral recombinant lactobacilli vaccines |
| CN101291691A (en) * | 2003-08-28 | 2008-10-22 | 免疫反应公司 | Immunogenic HIV compositions and related methods |
Non-Patent Citations (1)
| Title |
|---|
| GERALD K. CHEGE等: "A prime–boost immunisation regimen using recombinant BCG and Pr55gag virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons", <VACCINE>, vol. 27, 9 June 2009 (2009-06-09), pages 4857 - 4866, XP 026321684, DOI: doi:10.1016/j.vaccine.2009.05.064 * |
Also Published As
| Publication number | Publication date |
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| CN107899009B (en) | 2021-09-17 |
| ZA201307390B (en) | 2014-05-28 |
| CN103732248B (en) | 2018-08-28 |
| CN107899009A (en) | 2018-04-13 |
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