WO2014140938A2 - Méthodes immunologiques - Google Patents

Méthodes immunologiques Download PDF

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Publication number
WO2014140938A2
WO2014140938A2 PCT/IB2014/001268 IB2014001268W WO2014140938A2 WO 2014140938 A2 WO2014140938 A2 WO 2014140938A2 IB 2014001268 W IB2014001268 W IB 2014001268W WO 2014140938 A2 WO2014140938 A2 WO 2014140938A2
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cells
antigen
agent
regulatory
inhibits
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PCT/IB2014/001268
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WO2014140938A3 (fr
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Giuseppe Pantaleo
Sven LETOURNEAU
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Centre Hospitalier Universitaire Vaudois
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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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • This disclosure relates to reagent and methods for breaking peripheral immunological tolerance.
  • the reagents and methods described herein provide for improved immune responses by, for example, inducing the proliferation and / or activity of antigen-specific follicular helper T cells (e.g., CXCR5 PD- 1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans), antigen-specific memory B cells (e.g., B220 + IgG + cells in mice, CD19 CD27 CD38 + in humans), and / or the production of antibodies exhibiting increased avidity (as compared to those conventionally induced) against antigens of infectious agents.
  • antigen-specific follicular helper T cells e.g., CXCR5 PD- 1 + CD4 + T cells
  • antigen-specific germinal center B cells e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans
  • Figure 1 Depletion of FoxP3 + T regulatory cells upon PC61 (anti-CD25 antibody) treatment.
  • A Representative flow cytomerty profiles.
  • B Mean numbers +SEM of CD4 + FoxP3 + regulatory T cell subsets.
  • Figure 2. T cell responses after prime -boost vaccination using recombinant DNA prime and NYVAC boost with co-administration of PC61 (anti-CD25 antibody) (A, B: total responses; C, D: responses against individual peptide pools).
  • FIG. 1 B cell responses after prime-boost vaccination using recombinant DNA prime and NYVAC boost with co-administration of PC61 (anti-CD25 antibody) (A, B: ELISPOT; C, D: ELISA).
  • FIG. Follicular helper T cells and germinal center B cells expansion after prime- boost vaccination using recombinant DNA prime and NYVAC boost with co-administration of PC61 (anti-CD25 antibody) (A, B: Follicular helper T cells; C: B cells).
  • PC61 anti-CD25 antibody
  • Figure 5 Avidity of sera after prime-boost vaccination using recombinant DNA prime and NYVAC boost with co-administration of PC61 (anti-CD25 antibody).
  • the methods provide for inducing or enhancing the proliferation and / or activity of cells having specificity for an antigen, the cells being antigen- specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans), antigen- specific memory B cells (e.g., B220 + IgG + cells in mice, CD19 + CD27 + CD38 + in humans), and / or increasing the amount and / or avidity of antibodies against the antigen by administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells to a host.
  • Other embodiments are described herein and / or would be readily ascertainable therefrom by one of ordinary skill in the art.
  • This disclosure relates to methods for transiently breaking peripheral immunological tolerance in host such as a mammal (e.g., a human being).
  • the methods provide for inducing or enhancing the proliferation and / or activity of cells having specificity for an antigen, the cells being antigen-specific follicular helper T cells (e.g., CXCR5 PD-1 CD4 T cells), antigen-specific germinal center B cells (e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans), antigen-specific memory B cells (e.g., B220 + IgG + cells in mice, CD19 CD27 CD38 + in humans) by administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells to a host.
  • an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells to a host.
  • the methods provide for, or further provide for, inducing antibodies having increased avidity for an antigen by administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells, the increased avidity being relative to antibodies induced by an immunological composition administered without the agent that inhibits the function of regulatory T cells.
  • increased amounts of antibody may also be produced.
  • the immunological composition and the agent may be administered simultaneously. In some embodiments, the immunological composition and the agent are administered separately.
  • Certain embodiments involve the administration of at least two forms of an antigen (e.g., a peptide / polypeptide comprising an antigen and a nucleic acid encoding the antigen), the at least two forms being administered separately or together.
  • the different forms may be administered in a prime-boost format.
  • the agent that inhibits the function of regulatory T cells may be administered with one or both forms of the antigen (e.g., with at least one priming and at least boosting dose).
  • the immune response induced and / or enhanced by the methods described herein may be protective, therapeutic and / or prophylactic.
  • the immune response may also provide antibodies that may be neutralizing with respect to an infective agent.
  • the immunological composition(s) represent a vaccine.
  • the agent that inhibits the function of regulatory T cells in a host may be any agent (e.g., compound, drug, or the like) that sufficiently inhibits the activity and / or proliferation of a population of regulatory T cells ("T regs") such that the proliferation and / or activity of immune cells having specificity for an antigen contained within an immunological composition administered to the host, the cells being antigen-specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 + GL-7 + CD95 + cells in mice, CD19 CD27 CD38 in humans), antigen-specific memory B cells (e.g., B220 IgG cells in mice, CD19 CD27 CD38 + in humans) is enhanced, and / or such that the immune cells are released from any suppressive activity placed upon the same by such regulatory T cells.
  • T regs regulatory T cells
  • the agent that inhibits the function of regulatory T cells may be an antibody against CD25 (which may also be known as interleukin-2 receptor alpha (IL-2Ra), Tac) (e.g., anti-human CD25 (e.g., produced by any of the clones known in the art as ACT-1, BC96 (BioLegend ® Cat. No. 302601), CD25-3G10 (Life Technologies Cat. No. MHCD2505), MEM-181 (AbD serotec Cat. No.
  • CD25 which may also be known as interleukin-2 receptor alpha (IL-2Ra), Tac
  • IL-2Ra interleukin-2 receptor alpha
  • Tac anti-human CD25 (e.g., produced by any of the clones known in the art as ACT-1, BC96 (BioLegend ® Cat. No. 302601), CD25-3G10 (Life Technologies Cat. No. MHCD2505), MEM-181 (AbD serotec Cat. No.
  • Anti-CTLA-4 antibodies may also be suitable (e.g, ipilmumab (also known as MDX-010, MDX-101, and / or Yervoy (Medarex, Bristol-Myers Squibb)), Tremelimumab (also known as ticilimumab, CP- 675,206 (Pfizer, Inc., Medlmmune), among others).
  • the agent may also be an antineoplastic agent such as Dinileukin diftitox (trade name Ontak ® (Eisai, Inc.)) Other agents available to those of ordinary skill in the art may also be used.
  • an antigen may be a moiety (e.g., polypeptide, peptide or nucleic acid) comprising an epitope that induces or enhances the immune response of a host to whom or to which the immunogen is administered.
  • An immune response may be induced or enhanced by either increasing or decreasing the frequency, amount, or half-life of a particular immune modulator (e.g, the expression of a cytokine, chemokine, co-stimulatory molecule).
  • the immune response is typically directed against a cell or organism expressing the epitope and / or antigen.
  • an immune response may result from expression of an antigen in a host following administration of a nucleic acid vector encoding the immunogen to the host.
  • the immune response may result in one or more of an effect (e.g., proliferation and / or activity of antigen-specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 GL-7 CD95 cells in mice, CD19 CD27 CD38 in humans), antigen-specific memory B cells (e.g., B220 IgG cells in mice, CD19 CD27 CD38 in humans)).
  • an effect e.g., proliferation and / or activity of antigen- specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 GL-7 CD95 cells in mice, CD19 CD27 CD38 in humans), antigen- specific memory B cells (e.g., B220 IgG cells in mice, CD19 CD27 CD38 in humans)).
  • the immune response may involve, effect, or be detected in proliferation and / or activity of antigen-specific follicular helper T cells (e.g., CXCR5 PD-1 CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans), antigen-specific memory B cells (e.g., B220 + IgG + cells in mice, CD19 + CD27 + CD38 + in humans).
  • the immune response may involve, effect, or be detected in adaptive immune cells including, for example, lymphocytes (e.g., T cells and / or B cells).
  • the immune response may also be observed by detecting such involvement or effects including, for example, the presence, absence, or altered (e.g., increased or decreased) expression or activity of one or more immunomodulators such as a hormone, cytokine, interleukin (e.g., any of IL-1 through IL-35), interferon (e.g., any of IFN-I (IFN-a, IFN- ⁇ , IFN- ⁇ , IFN-K, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), IFN-II (e.g., IFN- ⁇ ), IFN-III (IFN- ⁇ , IFN- 2, IFN- 3)), chemokine (e.g., any CC cytokine (e.g., any of CCL1 through CCL28), any CXC chemokine (e.g., any of CXCL1 through CXCL24), Mipla), any C chemokine (e.g., XCL1, XCL2), any CX3C
  • the presence, absence or altered expression may be detected within cells of interest or near those cells (e.g., within a cell culture supernatant, nearby cell or tissue in vitro or in vivo, and / or in blood or plasma).
  • Administration of the immunogen may induce (e.g., stimulate a de novo or previously undetected response), or enhance or suppress an existing response against the immunogen by, for example, causing an increased antibody response (e.g., amount of antibody, increased affinity / avidity) or an increased cellular response (e.g., increased number of activated T cells, increased affinity / avidity of T cell receptors).
  • the immune response may be protective, meaning that the immune response may be capable of preventing initiation or continued infection of or growth within a host and / or by eliminating an agent (e.g., a causative agent, such as HIV) from the host.
  • an agent e.g., a causative agent, such as HIV
  • antigen-specific follicular helper T cells e.g., CXCR5 + PD-1 + CD4 + T cells
  • antigen-specific germinal center B cells e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans
  • antigen-specific memory B cells e.g., B220 + IgG + cells in mice, CD19 + CD27 + CD38 + in humans
  • amount and / or avidity of the antibodies produced may be determined using any of several currently-available techniques.
  • ex-vivo ELISPOT IFN- ⁇ assays may be conducted on fresh splenocytes as described previously (Miyahira Y et al, J Immunol Methods, 1995).
  • Antibody-secreting cells may be detected using standard techniques. For instance, splenocytes of animals to which an immunological composition(s) was previously administered may be restimulated in vitro for a sufficient period of time (e.g,. 48h) with one or more polyclonal activators (e.g., mIL-2 and R848) subsequently cultivated for a sufficient period of time (e.g., overnight) in anti-IgG-coated ELISPOT plates. ASCs may then be revealed using biotinylated antigen (e.g., biotinylated gpl40 as in the Examples) and streptavidin-conjugated horseradish peroxidase using standard techniques.
  • biotinylated antigen e.g., biotinyl
  • Intracellular cytokine staining techniques may also be used. For instance, cells may be restimulated with polyclonal activatiors (e.g., PMA and / or ionomycin (Sigma)) for a sufficient period of time (e.g., 90 min) followed by the addition of GolgiStop (BD Biosciences) and a further incubation period for a sufficient period of time (e.g., 5h).
  • polyclonal activatiors e.g., PMA and / or ionomycin (Sigma)
  • GolgiStop BD Biosciences
  • Cells may then be washed and stained with appropriate antibodies (e.g., anti-CD4-Alexa 405 (Invitrogen), anti-CD3- PerCP-Cy5.5, anti-CXCR5-APC, and / or -PD-1-FITC (BD Biosciences)). Cells may then be washed again, permeabilized (e.g., with Cytofix/Cytoperm (BD Biosciences)), and washed once more (e.g., with Perm/Wash buffer (BD Biosciences)). The cells may then be stained with an appropriate reagent (e.g., anti-IL-21-PE (R&D Sytems)).
  • appropriate antibodies e.g., anti-CD4-Alexa 405 (Invitrogen), anti-CD3- PerCP-Cy5.5, anti-CXCR5-APC, and / or -PD-1-FITC (BD Biosciences)
  • Cells may then be washed again, permeabilized (e.g.,
  • Cells may then be washed again, fixed (e.g., using Cell Fix (BD Biosciences)) and stored at an appropriate temperature (e.g., 4°C) until analysis.
  • a FoxP3 staining kit e.g., available from eBioscience
  • cells may be incubated under appropriate conditions (e.g., 30 min at 4°C in FACS wash buffer (PBS, 4% FCS, 5mM EDTA)) with an appropriate reagent (e.g., biotinylated gpl40 at 10 ⁇ g/ml).
  • Cells may then be washed and stained with one or more appropriate B cell detecting agents (e.g., anti-B220-Alexa 405 (Invitrogen), -GL-7-FITC, -CD95-PE and streptavidin-PerCP- Cy5.5 (all BD Biosciences)) under appropriate conditions (e.g., 15 min at 4°C).
  • B cell detecting agents e.g., anti-B220-Alexa 405 (Invitrogen), -GL-7-FITC, -CD95-PE and streptavidin-PerCP- Cy5.5 (all BD Biosciences)
  • appropriate conditions e.g., 15 min at 4°C
  • Cells may then be fixed (e.g., using Cell Fix (BD Biosciences)) and stored under appropriate conditions (e.g., 4°C) until analysis.
  • Stained cells may be detected using the appropriate equipment (e.g., LSRII cytometer (BD Biosciences)) and analytical software (e.g., FlowJ
  • ELISA may also be carried out using standard techniques.
  • antigen(s) e.g., various recombinant gpl40 proteins for HIV studies
  • plates e.g., Maxisorp plates (Nunc)
  • an appropriate concentration e.g., 2 ⁇ g/ml
  • serial dilutions of serum from control and / or immunized hosts applied to the plates.
  • Detection may be performed with an appropriate detection agent / system (e.g., secondary biotinylated goat anti-mouse IgG antibody (Sigma) and streptavidin-conjugated horseradish peroxidase).
  • TMB TMB (BD Biosciences) may be used as a substrate and the reaction may be stopped by addition of an appropriate reagent (e.g., 50 ⁇ 1 of 2 M H 2 SO 4 ). Optical densities may be read (e.g., at 450 nm) and results may be expressed as endpoint titers.
  • an appropriate reagent e.g., 50 ⁇ 1 of 2 M H 2 SO 4 .
  • Optical densities may be read (e.g., at 450 nm) and results may be expressed as endpoint titers.
  • plates may be coated with an appropriate amount of antigen (e.g., 2 ⁇ g/ml), and a saturating dilution of serum (as previously determined by ELISA) combined with serial dilutions of sodium thiocyanate (Sigma) may be added. The concentration of thiocyanate at which 50% of the bound antibodies are eluted may then be calculated and designated as the antibody avidity index.
  • test / experimental and control assays may be compared to determine whether a particular value is increased and / or decreased as a result of a particular immunization strategy. For example, whether an increased amount of antibody having specificity for the antigen is produced may be determined by comparing the amount of antibody detected following administration of the immunological composition with the agent that inhibits the function of regulatory T cells to that detected following administration of the immunological composition without the agent that inhibits the function of regulatory T cells.
  • the assay systems described above are merely exemplary and other assay systems may also be useful, as may be determined by one of ordinary skill in the art.
  • test / experimental and control assays may be compared to determine whether a particular parameter being measured (e.g., proliferation and / or activity of antigen- specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans), antigen- specific memory B cells (e.g., B220 IgG cells in mice, CD19 CD27 CD38 in humans), and /or the amount and / or avidity of antibodies) has changed (e.g., increased or decreased) as a result of a particular immunization / treatment strategy.
  • a particular parameter being measured e.g., proliferation and / or activity of antigen- specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g.
  • whether an increased amount of antibody having specificity for an antigen is produced may be determined by comparing the amount of antibody detected following administration of the immunological composition with the agent that inhibits the function of regulatory T cells to that detected following administration of the immunological composition without the agent that inhibits the function of regulatory T cells.
  • the difference between any one or more parameters may be measured at any appropriate time after the immunization and / or treatment begins. For instance, measurements may be made at any of day 0 (e.g., before administration of an immunological composition and / or agent that inhibits the function of regulatory T cells), day 1 , day 5, day 14, day 21 , day 28, day 35, day 41 , day 49, day 56, day 63, day 70, day 78, and / or day 84, for example. Data is typically compared between treatment regimens using measurements made on a particular day under similar conditions.
  • Data generated using such techniques may be analysed for statistical significance using standard statistical tools available to those of ordinary skill in the art.
  • the Examples describe the use of GraphPad Prism 6 statistical software (GraphPad Software Inc., USA), Mann- Whitney U tests, and / or Kruskal-Wallis ANOVA with Dunn's test (for multiple sample comparison).
  • the Examples also used a value of p ⁇ 0.05 as being statistically significant. Other tests and p values may also be used to determine statistical significance.
  • a statistically significant value that may be relied upon when comparing immune responses is p ⁇ 0.05, p ⁇ 0.04, p ⁇ 0.03, p ⁇ 0.02, p ⁇ 0.01 , p ⁇ 0.005, p ⁇ 0.001 , p ⁇ 0.0005, or pO.0001 , for example.
  • compositions described herein may include one or more immunogen(s) from a single source or multiple sources.
  • immunogens may also be derived from or direct an immune response against one or more viruses (e.g., viral target antigen(s)) including, for example, a dsDNA virus (e.g.
  • adenovirus adenovirus
  • herpesvirus Epstein-Barr virus
  • herpes simplex type 1 herpes simplex type 2
  • human herpes virus simplex type 8 human cytomegalovirus, varicella- zoster virus, poxvirus
  • ssDNA virus e.g., parvovirus, papillomavirus (e.g., El , E2, E3, E4, E5, E6, E7, E8, BPVl , BPV2, BPV3, BPV4, BPV5 and BPV6 (In Papillomavirus and Human Cancer, edited by H. Pfister (CRC Press, Inc. 1990); Lancaster et al, Cancer Metast. Rev. pp.
  • papillomavirus e.g., El , E2, E3, E4, E5, E6, E7, E8, BPVl , BPV2, BPV3, BPV4, BPV5 and BPV6
  • dsRNA viruses e.g., reovirus
  • (+)ssRNA viruses e.g., picornavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus
  • (+)ssRNA viruses e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rhabdovirus, rabies virus
  • ssRNA-RT viruses e.g.
  • HIV human immunodeficiency virus
  • dsDNA- RT viruses e.g. hepadnavirus, hepatitis B.
  • Immunogens may also be derived from other viruses not listed above but available to one of skill in the art.
  • immunogens may be selected from any HIV isolate.
  • HIV isolates are now classified into discrete genetic subtypes.
  • HIV-1 is known to comprise at least ten subtypes (Al , A2, A3, A4, B, C, D, E, Fl , F2, G, H, J and K) (Taylor et al, NEJM, 359(18): 1965-1966 (2008)).
  • HIV-2 is known to include at least five subtypes (A, B, C, D, and E).
  • Subtype B has been associated with the HIV epidemic in homosexual men and intravenous drug users worldwide.
  • Most HIV-1 immunogens, laboratory adapted isolates, reagents and mapped epitopes belong to subtype B.
  • HIV-1 subtype B In sub-Saharan Africa, India and China, areas where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small minority of infections, and subtype HIV-1 C appears to be the most common infecting subtype. Thus, in certain embodiments, it may be preferable to select immunogens from HIV-1 subtypes B and / or C. It may be desirable to include immunogens from multiple HIV subtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1 and HIV-2 subtypes) in a single immunological composition.
  • multiple HIV subtypes e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1 and HIV-2 subtypes
  • Suitable HIV immunogens include ENV, GAG, POL, NEF, as well as variants, derivatives, and fusion proteins thereof, as described by, for example, Gomez et al. Vaccine, Vol. 25, pp. 1969-1992 (2007).
  • Immunogens may also be derived from or direct an immune response against one or more bacterial species (spp.) (e.g., bacterial target antigen(s)) including, for example, Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp.
  • Bacillus spp. e.g., Bacillus anthracis
  • Bordetella spp. e.g., Bordetella pertussis
  • Borrelia spp. e.g., Borrelia burgdorf
  • Clostridium spp. e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani
  • Corynebacterium spp. e.g., Corynebacterium diptheriae
  • Enterococcus spp. e.g., Enterococcus faecalis, enterococcus faecum
  • Escherichia spp. e.g., Escherichia coli
  • Haemophilus spp. e.g., Haemophilus influenza
  • Helicobacter spp. e.g., Helicobacter pylori
  • Legionella spp. e.g., Legionella pneumophila
  • Leptospira spp. e.g., Leptospira interrogans
  • Listeria spp. e.g., Listeria monocytogenes
  • Mycobacterium spp. e.g., Mycobacterium leprae, Mycobacterium tuberculosis
  • Mycoplasma spp. e.g., Mycoplasma pneumoniae
  • Neisseria gonorrhea Neisseria meningitidis
  • Pseudomonas spp. e.g., Pseudomonas aeruginosa
  • Rickettsia spp. e.g., Rickettsia rickettsii
  • Salmonella spp. e.g., Salmonella typhi, Salmonella typhinurium
  • Shigella spp. e.g., Shigella sonnei
  • Immunogens may also be derived from or direct the immune response against other bacterial species not listed above but available to one of skill in the art.
  • Immunogens may also be derived from or direct an immune response against one or more parasitic organisms (spp.) (e.g., parasite target antigen(s)) including, for example, Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E.
  • parasitic organisms e.g., parasite target antigen(s)
  • Ancylostoma spp. e.g., A. duodenale
  • Anisakis spp. Ascaris lumbricoides
  • Balantidium coli Ces
  • Fasciola spp. e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni
  • Fasciolopsis buski Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M.
  • Necator americanus Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S.
  • Immunogens may also be derived from or direct the immune response against other parasitic organisms not listed above but available to one of skill in the art.
  • Immunogens may also be derived from or direct the immune response against tumor target antigens (e.g., tumor target antigens).
  • tumor target antigen may include both tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), where a cancerous cell is the source of the antigen.
  • TSA tumor-associated antigens
  • a TA may be an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cells or an antigen that is expressed on normal cells during fetal development.
  • a TSA is typically an antigen that is unique to tumor cells and is not expressed on normal cells.
  • TAs are typically classified into five categories according to their expression pattern, function, or genetic origin: cancer-testis (CT) antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens (i.e., Melan A/MART- 1, tyrosinase, gplOO); mutational antigens (i.e., MUM-1, p53, CDK-4); overexpressed 'self antigens (i.e., HER-2/neu, p53); and, viral antigens (i.e., HPV, EBV).
  • CT cancer-testis
  • MAGE MAGE
  • NY-ESO-1 melanocyte differentiation antigens
  • mutational antigens i.e., MUM-1, p53, CDK-4
  • overexpressed 'self antigens i.e., HER-2/neu, p53
  • viral antigens i.e., HPV, EBV
  • Suitable TAs include, for example, gplOO (Cox et al, Science, 264:716-719 (1994)), MART- 1 /Melan A (Kawakami et al, J. Exp. Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al, J. Exp. Med., 186: 1131-1140 (1996)), tyrosinase (Wolfel et al, Eur. J. Immunol, 24:759-764 (1994)), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al, J.
  • MAGE family antigens i.e., MAGE-1, 2,3,4,6, and 12; Van der Bruggen et al, Science, 254: 1643-1647 (1991); U.S. Pat. No. 6,235,525)
  • BAGE family antigens Boel et al, Immunity, 2: 167-175 (1995)
  • GAGE family antigens i.e., GAGE-1,2; Van den Eynde et al, J. Exp. Med., 182:689-698 (1995); U.S. Pat. No.
  • RAGE family antigens i.e., RAGE- 1; Gaugler et at., Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N- acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med., 183: 1173-1183 (1996)), pl5 (Robbins et al, J. Immunol. 154:5944-5950 (1995)), ⁇ -catenin (Robbins et al, J. Exp. Med., 183: 1185-1192 (1996)), MUM-1 (Coulie et al, Proc. Natl.
  • pl85 HER2/neu pl85 HER2/neu (erb-Bl; Fisk et al, J. Exp. Med., 181 :2109-2117 (1995)), epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29: 1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al, J. Natl. Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos.
  • carcinoma-associated mutated mucins i.e., MUC-1 gene products; Jerome et al, J. Immunol, 151 : 1654-1662 (1993)
  • EBNA gene products of EBV i.e., EBNA-1; Rickinson et al, Cancer Surveys, 13:53-80 (1992)
  • E7, E6 proteins of human papillomavirus Ressing et al, J.
  • Immunogens may also be derived from or direct the immune response against include TAs not listed above but available to one of skill in the art.
  • the antigen may be administered to the host in any suitable form (e.g., polypeptide, peptide, as an expressible nucleic acid).
  • Expression vectors may be used.
  • Exemplary expression vectors include, for instance, retrovirus (e.g., Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV)), adenovirus (as described by, for example, Rosenfeld, M., et al, 1991, Science, 252 (5004): 431-4; Crystal, R., et al, 1994, Nat.
  • retrovirus e.g., Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RS
  • vaccinia NYVAC
  • NYVAC U.S. Pat. Nos. 5,364,773; 5,494,807; and 6,265,189; ATCC VR-2559, VR- 2558, VR-2557, VR-2556, ATCC-979
  • ALVAC(2) e.g., U.S. Pat. Nos. 6,130,066 and 7,473,536
  • TROVAC ATCC 2553
  • Non-viral plamid vectors may also be suitable in certain embodiments. Plasmid vectors may also be suitable in certain embodiments. Preferred plasmid vectors are compatible with bacterial, insect, and / or mammalian host cells.
  • Such vectors include, for example, PCR- II, PCR3, and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La JoUa, CA), pET15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBACII, Invitrogen), pDSR-alpha (pet pub. No.
  • telomeres a high copy number cole 1 -based phagemid, Stratagene Cloning Systems, La Jolla, CA
  • PCR cloning plasmids designed for cloning Taq-amp lifted PCR products e.g. , TopoTM TA cloning ® kit, PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, CA.
  • Bacterial vectors may also be used such as, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille calmette guerin (BCG), and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376).
  • BCG Bacille calmette guerin
  • Streptococcus see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376).
  • Many other non-viral plasmid expression vectors and systems are known in the art and may be used.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo.
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et ah, 1981, Trends Biochem. Sci., 6: 77).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the immunological composition may comprise a naked recombinant DNA molecule encoding at least one antigen and a recombinant virus encoding at least one antigen (the same or different antigen).
  • the antigen of the naked recombinant DNA molecule and the recombinant virus typically share at least one common epitope.
  • the naked recombinant DNA molecule and the recombinant virus may each be administered with the agent that inhibits the function of regulatory T cells.
  • either the naked recombinant DNA molecule or the recombinant virus may be administered with the agent that inhibits the function of regulatory T cells.
  • the time period between administration of the naked recombinant DNA molecule and the agent that inhibits the function of regulatory T cells and administration of the recombinant virus and the agent that inhibits the function of regulatory T cells is about 7, about 14, or about 21 days.
  • the administrations may be separated by about any of 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, 5 years, or any time period before, after, and / or between any of these time periods.
  • Other embodiments of such methods may also be appropriate as could be readily determined by one of ordinary skill in the art.
  • the antigens / immunogens may also be combined with one or more pharmaceutically acceptable carriers prior to administration to a host (e.g., as an immunological composition and / or vaccine).
  • a pharmaceutically acceptable carrier is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Suitable pharmaceutical carriers and their formulations are described in, for example, Remington 's: The Science and Practice of Pharmacy, 21 st Edition, David B.
  • a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically- acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to about 7.5.
  • Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing polypeptides or fragments thereof. Matrices may be in the form of shaped articles, e.g., films, liposomes or microparticles.
  • Carriers are those suitable for administration of polypeptides and / or fragments thereof to humans or other subjects.
  • the compositions may also include carriers, thickeners, diluents, buffers, preservatives, surface active agents, adjuvants, immunostimulants, in addition to the immunogenic polypeptide.
  • the compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents and anesthetics. Adjuvants may also be included to stimulate or enhance the immune response.
  • Non-limiting examples of suitable classes of adjuvants include those of the gel-type (i.e., aluminum hydroxide/phosphate ("alum adjuvants"), calcium phosphate, microbial origin (muramyl dipeptide (MDP)), bacterial exotoxins (cholera toxin (CT), native cholera toxin subunit B (CTB), E. coli labile toxin (LT), pertussis toxin (PT), CpG oligonucleotides, BCG sequences, tetanus toxoid, monophosphoryl lipid A (MPLA) of, for example, E.
  • gel-type i.e., aluminum hydroxide/phosphate (“alum adjuvants”), calcium phosphate, microbial origin (muramyl dipeptide (MDP)), bacterial exotoxins (cholera toxin (CT), native cholera toxin subunit B (CTB), E. coli labile toxin (LT), pertussis
  • coli Salmonella Minnesota, Salmonella typhimurium, or Shigella exseri
  • particulate adjuvants biodegradable, polymer microspheres
  • ISCOMs immunostimulatory complexes
  • FIA oil-emulsion and surfactant- based adjuvants
  • MF59, SAF microfluidized emulsions
  • QS-21 saponins
  • synthetic (muramyl peptide derivatives murabutide, threony-MDP
  • nonionic block copolymers L121
  • PCCP polyphosphazene
  • synthetic polynucleotides poly A :U, poly I :C
  • thalidomide derivatives CC-4407/ACTIMID
  • RH3-ligand or polylactide glycolide (PLGA) microspheres, among others.
  • Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.
  • Suitable mutants or variants of adjuvants are described, e.g., in WO 95/17211 (Arg-7- Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).
  • Additional LT mutants that may used include, for example, Ser-63-Lys, Ala-69- Gly,Glu-l 10-Asp, and Glu-112-Asp mutants.
  • kits for detecting a relevant immune response using a biological fluid by detecting the induction and / or enhancment of the proliferation and / or activity of cells having specificity for an antigen, the cells being antigen- specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 + GL-7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans), antigen- specific memory B cells (e.g., B220 IgG cells in mice, CD19 CD27 CD38 in humans), and / or antibodies (e.g., of increased avidity for the antigen) in the biological sample.
  • a biological fluid e.g., culture specimen, blood
  • One or more diagnostic kit components may be provided in a suitable container such as a vial to provide protection from the external environment.
  • a kit for detecting the presence and / or activity of a particular cell type following administration of one or more immunological compositions and one or more agents agent that inhibits the function of regulatory T cells is provided (e.g., secondary reagents such as labeled anti-antibody antibodies).
  • An exemplary kit may therefore comprise: (i) one or more control cell and / or biological samples; and, (ii) a system for detecting the induction and / or enhancement of the proliferation and / or activity of cells having specificity for an antigen, the cells being antigen-specific follicular helper T cells (e.g., CXCR5 + PD-1 + CD4 + T cells), antigen-specific germinal center B cells (e.g., B220 + GL- 7 + CD95 + cells in mice, CD19 + CD27 + CD38 + in humans), antigen-specific memory B cells (e.g., B220 + IgG + cells in mice, CD19 + CD27 + CD38 + in humans), and / or antibodies (e.g., of increased avidity for the antigen).
  • antigen-specific follicular helper T cells e.g., CXCR5 + PD-1 + CD4 + T cells
  • antigen-specific germinal center B cells e.g., B220 + GL
  • the kit may comprise one or more cells and / or antigens free in solution and / or immobilized on a solid support, such as a magnetic bead, tube, microplate well, or chip.
  • a solid matrix comprising one or more polypeptides and / or nucleic acids adsorbed thereto is provided.
  • the kit may further comprise a binding agent (e.g., antibody) as a detection reagent.
  • the detection reagent and may be free in solution or may be immobilized on a solid support, such as a magnetic bead, tube, microplate well, or chip.
  • the antibody-binding molecule, polypeptide, and / or nucleic acid may be also labeled with a detectable label, for example a fluorescent or chromogenic label or a binding moiety such as biotin.
  • the kit may further comprise detection reagents such as a substrate, for example a chromogenic, fluorescent or chemiluminescent substrate, which reacts with the label, or with molecules, such as enzyme conjugates, which bind to the label, and / or produce a signal.
  • the detection reagents may further comprise buffer solutions, wash solutions, and other useful reagents.
  • the kit may also comprise one or both of an apparatus for handling and/or storing the sample obtained from the individual and an apparatus for obtaining the sample from the individual (e.g., a needle, lancet, and collection tube or vessel).
  • the kit may also include instructions for use.
  • Other embodiments of kits are also contemplated as would be understood by those of ordinary skill in the art.
  • this disclosure provides methods for inducing or enhancing the proliferation and / or activity of follicular helper T cells having specificity for an antigen by administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells to a host.
  • this disclosure provides methods for inducing or enhancing the proliferation and / or activity of germinal center B cells having specificity for an antigen by administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells to a host.
  • this disclosure provides methods for inducing or enhancing the proliferation and / or activity of B cell memory cells having specificity for an antigen by administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells to a host.
  • this disclosure provides methods for inducing or enhancing the proliferation and / or activity of cells having specificity for an antigen, the cells being follicular helper T cells, germinal center B cells, and B cell memory cells, the method comprising administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells to a host.
  • this disclosure provides methods for inducing antibodies having increased avidity for an antigen iericn a host, the method comprising administering to a host an immunological composition comprising the antigen and an agent that inhibits the function of regulatory T cells, the increased avidity being relative to antibodies induced by an immunological composition comprising administering without the agent that inhibits the function of regulatory T cells.
  • the methods may provide for the production of an increased amount of antibody having specificity for the antigen in a host when compared to the amount of antibody produced following administration of the immunological composition without the agent that inhibits the function of regulatory T cells.
  • the methods may comprise inducing antibodies having increased avidity for an antigen in a host relative to antibodies induced by an immunological composition administered without the agent that inhibits the function of regulatory T cells.
  • the antibodies having increased avidity, and / or being present in increased amounts may be present and / or detected a suitable time after administration of the immunological composition (e.g., about any of one, two, three, four, five, six, seven, eight weeks).
  • the immunological composition and the agent are administered simultaneously or separately (e.g., at the same or different time and / or physically together or physically apart from one another).
  • the immunological composition and the agent may be administered together in time (e.g., at about the time of vaccination such as, for instance, on the same day).
  • the immunological composition and the agent may be administered at different times (e.g., one or the other may be administered before or after the other).
  • the time between administrations may be significant (e.g., one or more days such as, for instance, about any of one, two, three, four, five, six, seven, ten or 14 days).
  • the agent may be administered before (e.g., significantly before) the immunological composition.
  • the immunological composition comprises at least two forms of an antigen (e.g., a peptide, polypeptide, nucleic acid encoding the same) that are administered separately or together (e.g., at the same or different time and / or physically together).
  • at least one form of the antigen is a peptide or polypeptide encoded by a nucleic acid molecule and at least one form of the antigen is a peptide or polypeptide.
  • the immunological composition may be administered in a prime-boost format. Certain embodiments include administration of at least two forms of antigen separately and the agent that inhibits the function of regulatory T cells may be administered with both forms of the antigen.
  • the agent that inhibits the function of regulatory T cells may be administered with at least one priming dose and at least one boosting dose.
  • the antigen may be an HIV antigen.
  • the immunological composition may comprise a naked recombinant DNA molecule encoding at least one HIV antigen and a recombinant virus encoding at least one HIV antigen.
  • the HIV antigen of the naked recombinant DNA molecule and the recombinant virus share at least one common epitope.
  • the agent that inhibits the function of regulatory T cells may be selected from the group consisting of an antibody against CD25, anti-human CD25, anti-mouse CD25, an antibody against CTLA-4, anti-CTLA-4, ipilmumab, Tremelimumab, an antineoplastic agent, and Dinileukin diftitox.
  • the resulting immune response is protective, therapeutic and / or prophylactic.
  • Antibodies resulting from administration of the immunological composition(s) may be neutralizing with respect to an infective agent.
  • the immunological composition(s) may be a vaccine.
  • a subject or a host is meant to be an individual.
  • the subject can include domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs) and birds.
  • livestock e.g., cattle, horses, pigs, sheep, and goats
  • laboratory animals e.g., mice, rabbits, rats, guinea pigs
  • the subject is a mammal such as a primate or a human.
  • Optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • the phrase optionally the composition can comprise a combination means that the composition may comprise a combination of different molecules or may not include a combination such that the description includes both the combination and the absence of the combination (i.e., individual members of the combination).
  • Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed.
  • prevent, preventing, and prevention are used herein in connection with a given treatment for a given condition (e.g., preventing infection by Streptococcus sp.), it is meant to convey that the treated patient either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment. These terms are not limited solely to a situation in which the patient experiences no aspect of the condition whatsoever.
  • a treatment will be said to have prevented the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the patient's experiencing fewer and/or milder symptoms of the condition than otherwise expected.
  • a treatment can "prevent" infection by resulting in the patient's displaying only mild overt symptoms of the infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
  • reduce, reducing, and reduction as used herein in connection with the risk of infection with a given treatment typically refers to a subject developing an infection more slowly or to a lesser degree as compared to a control or basal level of developing an infection in the absence of a treatment (e.g., administration or vaccination using polypeptides disclosed).
  • a reduction in the risk of infection may result in the patient's displaying only mild overt symptoms of the infection or delayed symptoms of infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
  • mice Six to eight week old female BALB/c mice (Charles River) were used in compliance with the University of Lausanne Institutional regulations and with the approval of the veterinarian authorities of the Swiss Canton of Vaud. Mice received a single intra-muscular (i.m.) injection in the tibialis anterior muscle of 100 ⁇ of recombinant DNA- vectored vaccine; followed 2 weeks later by an i.m. injection of lxlO 7 PFU of NYVAC-vectored vaccine expressing the same immunogen. For anti-CD25 mAb administration, 10( ⁇ g of PC61 were injected intra-peritoneally (i.p.) at the time of vaccination. Animals were sacrificed 4 and 12 weeks after priming and spleen was collected, as well as blood from a superficial vein. Popliteal and inguinal lymph nodes were collected when draining lymph nodes were analyzed.
  • Ex-vivo ELISPOT IFN- ⁇ assays were conducted on fresh splenocytes as described previously (Miyahira Y et al, J Immunol Methods, 1995). Antibody-secreting cells (ASC) were detected following the manufacturer's recommendations (Mabtech). Briefly, splenocytes were restimulated in vitro for 48h with polyclonal activators mIL-2 and R848, and subsequently cultivated over night in anti-IgG-coated ELISPOT plates. ASCs were revealed using biotinylated gpl40z M 96 and streptavidin-conjugated horseradish peroxidase.
  • cytokine staining For intracellular cytokine staining, cells were restimulated with polyclonal activatiors PMA and ionomycin (Sigma) for 90 min followed by the addition of GolgiStop (BD Biosciences) and a further incubation period of 5h. Cells were then washed and stained with anti-CD4-Alexa 405 (Invitrogen), -CD3-PerCP-Cy5.5, -CXCR5-APC and -PD-1-FITC (BD Biosciences).
  • Cells were then washed and stained with anti-B220-Alexa 405 (Invitrogen), -GL-7-FITC, -CD95-PE and streptavidin-PerCP-Cy5.5 (all BD Biosciences) for 15 min at 4°C. Cells were fixed with Cell Fix (BD Biosciences) and stored at 4°C until analysis. All stained cells were acquired on LSRII cytometer (BD Biosciences) and data was analyzed using Flow Jo Software (Three Star).
  • Optical densities were read at 450 nm, results are expressed as endpoint titers.
  • For measuring avidity plates were coated with 2 ⁇ g/ml of ZM96 gpl40, and a saturating dilution of serum (as previously determined by ELISA) combined with serial dilutions of sodium thiocyanate (Sigma) was added. The concentration of thiocyanate at which 50% of the bound antibodies are eluted was calculated and designated as the antibody avidity index.
  • T cell responses after vaccination with co-administration of PC61 are illustrated in Figure 2.
  • Groups of four to five BALB/c mice received a single intramuscular (i.m.) injection of recombinant DNA expressing HIV-1 clade C proteins Gag, Pol, Nef and Env; followed two weeks later by a booster i.m. injection of recombinant NYVAC expressing the same immunogen.
  • One group of mice also received an i.p. injection of 100 ⁇ g of anti-CD25 mAb with each vaccine administration.
  • FIG. 3 B cell responses after vaccination with co-administration of PC61 (anti-CD25 mAb) are illustrated in Figure 3.
  • Groups of 4 to 5 BALB/c mice received a single i.m. injection of recombinant DNA expressing HIV-1 clade C proteins Gag, Pol, Nef and Env; followed two weeks later by a booster i.m. injection of recombinant NYVAC expressing the same immunogen.
  • One group of mice also received an i.p. injection of 100 ⁇ g of anti-CD25 mAb with each vaccine administration.
  • FIGs. 3 A, 3C At 4 (Figs. 3A, 3C) and 12 (Figs. 3B, 3D) weeks after priming animals were sacrificed.
  • Anti-gpl40 antibody secretion by restimulated splenocytes was measured by ELISPOT (Figs. 3A, 3B). Columns represent mean anti-gpl40 antibody secreting cells (ASC) per million cells +SEM. Serum reactivity with gpl40 proteins from different isolates as indicated on the lower axis (clade specified in brackets) was assessed by ELISA (Figs. 3C, 3D). Columns represent mean end-dilution titers +SEM. The data is representative of three separate experiments. Statistical significance is indicated by "*" (P ⁇ 0.05). As shown therein, co-administration of PC61 significantly increases antigen-specific B cell frequency, antibody titers and antibody cross-reactivity following DNA/NYVAC immunization protocols in most animals at four weeks.
  • Figure 4 illustrates the expansion of follicular helper T cells and germinal center B cells upon PC61 (anti-CD25 mAb) co-treatment.
  • Groups of four to five BALB/c mice received a single i.m. injection of recombinant DNA expressing HIV-1 clade C proteins Gag, Pol, Nef and Env; followed two weeks later by a booster i.m. injection of recombinant NYVAC expressing the same immunogen.
  • One group of mice also received an i.p. injection of 10( ⁇ g of anti-CD25 mAb with each vaccine administration. Four weeks after priming, the animals were sacrificed and draining lymph nodes harvested.
  • Figure 5 demonstrates that the avidity of sera from immunized mice is increased when the immunogen is co-administered with PC61 (anti-CD25 mAb).
  • Groups of four to five BALB/c mice received a single i.m. injection of recombinant DNA expressing HIV-1 clade C proteins Gag, Pol, Nef and Env, followed two weeks later by a booster i.m. injection of recombinant NYVAC expressing the same immunogen.
  • One group of mice also received an i.p. injection of 100 ⁇ g of anti-CD25 mAb with each vaccine administration. At four weeks after priming animals were sacrificed. Avidity of sera was measured by ELISA, and expressed as mean avidity index +SEM.

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Abstract

La présente invention concerne un réactif et des méthodes permettant de rompre la tolérance immunologique périphérique.
PCT/IB2014/001268 2013-03-14 2014-03-14 Méthodes immunologiques WO2014140938A2 (fr)

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