Abstract
The invention pertains to an immunization scheme for inducing or amplifying an immune response involving Variant Surface Glycoproteins as carriers for antigenic structures against which the immune response is targeted. The invention is based on a specific priming and boosting schedule using the array presented and soluble VSG variants. Surprisingly, the immunization scheme of the invention elicits a strong and long-lasting antibody response in the immunized subject and therefore is applicable in vaccination approaches, immunotherapy and in the production of novel antibodies.
Claims
1. A method for eliciting an immune response, and/or for amplifying a pre-existing immune response, against an antigenic compound in a subject, comprising at least one priming-immunization step and at least one boosting-immunization step, wherein the at least one boosting-immunization step is performed after the at least one priming-immunization step, and wherein: (i) The at least one priming-immunization step comprises an immunization of the mammal with an array of immunization-carrier-fusions on a vesicle or bead, and wherein each immunization-carrier-fusion is composed of a carrier protein fused to the antigenic compound; and (ii) The at least one boosting-immunization step comprises an immunization of the mammal with the immunization-carrier-fusion used in (i) in a non-aggregated, preferably soluble form.
2. The method of claim 1, wherein the immunization-carrier-fusion is an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), wherein the eVSG comprises the antigenic compound covalently linked, optionally via a linker, to the N-terminus of the VSG carrier protein.
3. The method of claim 1, wherein the immunization-carrier-fusion has the following (covalent) structure from N- to C-terminus: antigenic compound, a sortagging donor sequence, a sortagging acceptor sequence, a linker, a VSG protein sequence.
4. The method of claim 1, wherein the antigenic compound is a disease associated antigen, for example a cancer antigen, an allergen or a viral immunogen, or is a dependency causing substance selected from (i) delta tetrahydrocannabinol (THC) or Synthetic cannabinoids, such as classical cannabinoids, non-classical cannabinoids, hybrid cannabinoids, aminoalkylindoles, and eicosanoids; for example Δ9-THC HU-210, (C8) CP 47,497, JWH-018, AM-2201 (Fluorinated JWH-018), UR-144, XLR-11 (Fluorinated UR-144), APICA, STS-135 (Fluorinated APICA). AB-PINACA, PB-22, 5F-PB-22 (Fluorinated PB-22); or (ii) methamphetamine and derivatives thereof such as 3,4-methylenedioxy-methamphetamine (MDMA)Ecstasy/Molly; or (iii) a synthetic cathinone like alpha-pyrrolidinopentiophenone (alpha-PVP); or (iv) an opioid including heroin, synthetic opioids such as fentanyl or related compounds such as carfentanyl, and other opioid pain relievers, such as oxycodone (OxyContin®), hydrocodone (Vicodin®), codeine, morphine, desomorphine (Krokodil); or (v) steroids (anabolic substances), or is nicotine.
5. The method of claim 1, to wherein the carrier protein is a VSG protein derived from Trypanosoma brucei.
6. The method of claim 1, wherein the vesicle or bead is coated with more than one immunization-particle fusions, preferably more than 10 immunization-particle fusions per particle.
7. The method of claim 1, wherein the immunization-carrier used in the method is a VSG protein, and wherein the VSG protein used as carrier in (i) and (ii) are derived from the same VSG protein type, for example the VSG in both (i) and (ii) are VSG3 of Trypanosoma brucei.
8. The method of claim 1, wherein the method comprises at least two priming-immunization steps, and wherein all of the priming immunization steps are performed prior to any of the boosting-immunization steps.
9. The method of claim 1, wherein the method comprises at least two boosting-immunization steps, and wherein all of the boosting immunization steps are performed after any of the priming-immunization steps.
10. The method of claim 1, wherein the subject is selected from an animal having an adaptive immune system, such as vertebrates such as birds (chicken), and mammals such as mouse, rabbit, camel, goat, rat, dog, cat, monkey, hamster or other mammals, or is a human, such as a human patient in need of an elicited or amplified immune response.
11. A non-therapeutic method for generating antibodies against an antigenic-compound, the method comprising performing in a non-human animal the method of claim 1, and isolating from the non-human animal antibodies or B-cells producing antibodies against the antigenic compound.
12. An antibody, or an antibody producing B-cell, generated and/or isolated according to a method of claim 11.
13. An immunization kit, the immunization kit comprising (a) an aggregate of immunization-carrier protein on a vesicle or bead as recited in claim 1, and (b) a non-aggregated, preferably soluble form, of the immunization-carrier protein of (a); wherein the immunization-carrier protein of (a) and (b) are capable to be fused to an antigenic compound to obtain an immunization-carrier-fusion recited in claim 1.
14. A pharmaceutical composition, comprising the immunization-carrier-fusion claim 1.
Description
BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES
[0128] The figures show:
[0129] FIG. 1. Map of plasmids used for VSG engineering. Sequences of pHH-VSG3.G4S-Hyg and pHH-ILTat1.24-G4S-Hyg plasmids are provided in SEQ ID NO: 6 and SEQ ID NO: 7 respectively. (A) pHH-VSG3.G4S-Hyg Plasmid Size: 7204 bp. VSG2-CTR (VSG2 Co-transposed Region): 429-878; VSG3.G4S (S317A): 879-2453; VSG2 3′-UTR (VSG2 3′-Untranslated Region): 2454-2533; Actin 5′-UTR (Actin 5′-Untranslated Region): 2727-2834; Hygro (Hygromycin Resistance Gene): 2854-3879; Aldolase 3′-UTR (Aldolase 3′-Untranslated Region): 3885-4033; Telomere Seed Sequences: 4715-4915; Ori (Bacterial Origin of Replication): 5385-5973; AmpR (Ampicillin Resistance Gene, Beta-lactamase): 6144-7004 (Reverse Complement); AmpR Promotor (Ampicillin Resistance Gene Promoter, Beta-Lactamase Promoter): 7005-7109 (Reverse Complement); (B) pHH-ILTat1.24-G4S-Hyg Plasmid Size: 7219 bp. VSG2-CTR (VSG2 Co-transposed Region): 429-878; ILTat1.24-G4S: 879-2468; VSG2 3′-UTR (VSG2 3′-Untranslated Region): 2469-2548; Actin 5′-UTR (Actin 5′-Untranslated Region): 2742-2849; Hygro (Hygromycin Resistance Gene): 2869-3894; Aldolase 3′-UTR (Aldolase 3′-Untranslated Region): 3900-4048; Telomere Seed Sequences: 4730-4930; Ori (Bacterial Origin of Replication): 5400-5988; AmpR (Ampicillin Resistance Gene, Beta-lactamase): 6159-7019 (Reverse Complement); AmpR Promotor (Ampicillin Resistance Gene Promoter, Beta-Lactamase Promoter): 7020-7124 (Reverse Complement). (C) The sequence of pPNSM2, importantly encoding “sortaggable VSG3” (synonymous with “VSG3-G4S (S317A)”) is provided in SEQ ID NO: 6. pPNSM2 Plasmid Size: 7060 bp. Key restriction sites for cloning and cell line generation are marked, with the bold sites representing the two options for linearization prior to transfection. The key elements of the plasmid are also marked, with the following coordinates in order: BES1 Co Transposed Region (1-1227, acting as a homologous region to facilitate genomic integration), Sortaggable VSG3 (1247-2821), VSG 3UTR (2828-2896), Actin 5′-UTR (3087-3193), Hygro (3219-4244, delineating the hygromycin resistance gene open reading frame), Aldolase 3′-UTR (4255-4395), ori (5020-5608, delineating the bacterial origin of replication on the bottom strand), AMP R (5779-6639, delineating the bacterial ampicillin resistance cassette on the bottom strand), and telomere seeds (6785-6985). Plasmids for VSG2 and ILtat1.2 can be generated based on the plasmids described for VSG3 above by replacing the sortaggable VSG3 by VSG2 or ILtat1.2.
[0130] FIG. 2. Crystallographic studies help to determine the accessibility of the VSG N-terminus to the sortase enzyme as illustrated using 5 examples (all published except VSG13). “N” denotes the location of the free N-terminus for each protein. The line denotes a roughly estimated general location of surface-exposed residues (the “top” of the VSG coat, what would be accessible to the sortase enzyme). While many VSGs can be engineered to accept tags through sortase-based conjugation (and the inventors have already done this for VSG2, VSG3 and ILTat1.24 as discussed later), not all can be (for example the N-terminus in VSG13 is far more buried and may not be sufficiently accessible). Structural biology presents a very useful pre-screening of VSGs to uncover many surface elements and other architectural features that inform the choices of VSGs (e.g., the discovery of the O-linked sugar on VSG3 that led the inventors to work with a specific serine to alanine mutant, S317A, to remove it from the surface).
[0131] FIG. 3. Knock-in strategy of sortaggable VSGs into the genome of Trypanosoma brucei. A to C show the genetic cloning strategy to express engineered VSG2, VSG3 and ILTat1.24 from the active expression site of endogenous VSG (replacing wild-type VSG2).
[0132] FIG. 4. Amino acid sequences of the sortaggable VSG proteins. A. Amino acid sequence of the sortaggable VSG3.G4S (S317A) protein. The signal peptide is underlined. The mature, sortaggable VSG3 is derived from a more antigenic mutant of wild-type VSG3 (S317A). VSG3-G4S will initiate with the di-Alanine (in bold and italic). This dipeptide will be the acceptor of the sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The extension of the N-terminus (by addition of the (G.sub.4S).sub.3 peptide linker, in bold), is crucial for the ability of sortase A to access the di-Alanine. B. Amino acid sequence of the sortaggable VSG2 protein (VSG2-1DK). The signal peptide is underlined. The mature, sortaggable VSG2-1DK will initiate with the di-Alanine (in bold and italic). This dipeptide will be the acceptor of the sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The extension of the N-terminus (by addition of a linker peptide consisting of a TEV protease cleavage site flanked by poly-Glycine, in bold) is crucial for the ability of sortase A to access the di-Alanine. C. Amino acid sequence of the sortaggable ILTat1.24 protein (ILTat1.24-G4S). The signal peptide is underlined. The mature, sortaggable ILTat1.24-G4S will initiate with tetra-Glycine (shown in bold and italic). This tetra-Glycine will be the acceptor of the Sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The extension of the VSG N-terminus (by addition of the (G4S)2 peptide linker, in bold), is crucial for the ability of Sortase A to access the tetra-Glycine.
[0133] FIG. 5. Wild-type T. brucei sheds its VSG coat upon death. A. Cartoon of the process showing that GPI-PLC is activated upon cell death. B. The location of GPI anchor cleavage site is shown. Cleavage of the GPI anchor releases (sheds) VSG from the coat and the coat-less trypanosome disintegrates through osmotic pressure (and consequently losing its immunogenic properties).
[0134] FIG. 6. Illustration of the overall method of sortagging the VSG coat of a Trypanosome. A. Visualization of a VSG protein homodimer embedded in the membrane of a Trypanosome via a glycophosphatidylinositol (GPI) anchor. Bottom: whole trypanosome. Top: Zoom on one VSG protein homodimer. B. Illustration of the sortagging reaction: modified VSG proteins (with an N-terminal di-Alanine), and small molecules (oval) linked to the sortase donor sequence LPSTGG, are covalently linked via a sortase reaction.
[0135] FIG. 7. Methods to detect Sortagging efficiency. A. Top image: Sortagging of VSG2-1DK (left) and VSG3-G4S (S317A) (right) detected via direct fluorescence (6-FAM). Fluorescent microscopic images of a T. brucei cell are shown at top (left: sortagged VSG2-1DK, right: sortagged VSG3-G4S). Bottom image: The 6-FAM sortagged VSGs were also analyzed by flow cytometry analysis using FACSCalibur. B. Sortagging of VGS2-1DK and VSG3-G4S (S317A) detected via FACS analysis of Trypanosomes using a monoclonal antibody against a small-molecule moiety (4-hydroxy-3-nitrophenylacetyl, abbreviated as NP here) followed by staining with an Allophycocyanin (APC)-conjugated mouse monoclonal IgG antibody (B1-8 clone, Abcam). C. A derivatized fentanyl hapten was chemically synthesized and conjugated to the N-terminus of a peptide containing a C-terminal sortase A donor sequence (fentanyl-GGGSLPSTGG, where fentanyl-conjugated compounds are alternatively denoted “Fen-” or “—Fen”). The peptide carrying the fentanyl hapten was conjugated to three different genetically modified VSGs (VSG2, VSG3 and ILTat1.24) using sortase A as described before. Chemical synthesis process of the fentanyl hapten has been described by M. D. Raleigh et al., J. Pharmacol. Exp. Ther. 368:282-291, 2019, and it has been adapted for sortase-mediated conjugation here. D. After sortase A-mediated conjugation of the fentanyl hapten to VSGs, a mouse monoclonal antibody against fentanyl (provided by M. Pravetoni, University of Minnesota) was conjugated to FITC using a kit (Abcam, ab102884) and used to stain the Sortagged VSGs followed by flow cytometry analysis using FACS-Calibur. Non-tagged VSGs were used as control for background staining. Below the graph: The mode and median of the data sets are shown. In both fentanyl and FAM conjugations, ILTat1.24 outperformed VSG2 and VSG3. Also, Sortagging efficiency of VSG3 was moderately higher than VSG2.
[0136] FIG. 8. Comparison of antibody responses to the small molecule 4-hydroxy-3-nitrophenyl acetyl (NP): Immunization with NP-labeled T. brucei vs. the “gold standard” hapten-carrier conjugate (i.e. NP-conjugated chicken gamma-globulin (NP-CGG) in Alum adjuvant). Five 6-8 weeks old female C57BL/6J mice per group were primed at day 0, 3 and 30 with intact VSG3(S317A), or VSG3-NP coats (i.e. U.V-irradiated intact T. brucei cells expressing sortaggable VSG3(S317A), either tagged or not tagged with NP hapten, without adjuvant) or with NP-CGG in Alum adjuvant. These mice received a soluble VSG3(S317A)-NP (in PBS, without adjuvant) booster at day 70 (or were boosted with soluble NP-CGG, in PBS without adjuvant, for the control group). A. The priming immunization with VSG-NP on intact trypanosomes followed by boosting with soluble VSG3(S317A)-NP, shows a clear IgG recall response and results in the generation of substantial IgG titers to the small molecule hapten NP. Titers were measured before and after boost and are shown at serum dilution 1:800. An anti-NP hapten monoclonal IgG (B1-8 clone, Abcam) was serially diluted (4-fold) to cover a range of concentrations from 10 μg/ml to 0.6 ng/ml. Immunization with the conjugated trypanosome coats results in high IgG titers to NP (average ˜500 μg/ml). Furthermore, the fact that soluble VSG3-NP (in PBS, without adjuvant) induced a secondary IgG response strongly indicates that immunization with NP-conjugated VSG 1.0 coats can induce a memory B cell response. While mean titers raised against NP-CGG in Alum are somewhat higher, boosting with soluble NP-CGG (in PBS, without adjuvant) did not induce a robust secondary response, which indicates lack of a memory B cell response after priming with NP-CGG in Alum. B. Immunization using VSG-NP on intact trypanosome coats followed by boosting with soluble VSG3(S317A) NP results in high affinity IgG (as defined by NP2/NP30 IgG titer ratios). Increase in NP2-BSA/NP30-BSA IgG ratio in VSG3-NP group indicates affinity maturation, a hallmark of memory B cell (recall) response. Immunization with the “gold standard” hapten-carrier conjugate (NP-CGG) in Alum provides no increase in affinity of anti-NP IgG antibodies. C. Immunization with VSG-NP on intact trypanosome coats followed by boosting with soluble VSG3(S317A)-NP also yields anti-VSG3 (anti-carrier) antibodies, but those are of a comparable magnitude (in μg/ml) to antisera raised to the NP hapten (as quantified by serial dilutions of an anti-VSG3 mouse monoclonal IgG (11D6 clone). Additional data demonstrate a lack of immunological cross-reactivity between the VSG2 and VSG3 carriers (not shown).
[0137] FIG. 9. Antibody responses to fentanyl elicited by Fen-labeled T. brucei achieve high titers and memory (recall). ELISA measurement of serum IgG against fentanyl hapten and VSG3 carrier protein are shown. A. A tetra-Glycine peptide carrying an N-terminal fentanyl hapten (Fen-G4) was synthesized as described before. The Fen-G4 peptide was conjugated to BSA as a heterologous carrier protein and used to coat 96-well ELISA plates at 10 μg/ml. Five 6-8 weeks old female C57BL/6J mice per group were primed at day 0 and 30 with intact VSG3(S317A), or VSG3(S317A)-Fent coats (i.e. U.V-irradiated intact T. brucei cells expressing sortaggable VSG3(S317A), either tagged or not tagged with Fen-hapten, without adjuvant) via subcutaneous injection. These mice then received a soluble VSG3(S317A)-Fent (in PBS, without adjuvant) booster at day 60. Serum samples tested included the pre-immune (day −4), 2 days before (day 58) and 8 days after (day 68) 1 boost with soluble VSG3(S317A)-fentanyl protein. Anti-fentanyl IgG in sera was detected using an anti-mouse HRP conjugate (1:3000) followed by addition of ABTS substrate and H.sub.2O.sub.2 prepared in citrate-phosphate buffer pH 4.2. Absorption of the samples were measured after 45 min at A405 nm using an ELISA reader (Tecan, Infinite M1000 Pro). B. A mouse monoclonal antibody against Fen-hapten was serially diluted to make a calibration curve in order to quantify IgG concentration in serum samples. Mean±standard deviation of 6 mice per group are shown. C. Similarly, 96-well plates were coated with FPLC-purified VSG3(S317A) protein at 5 μg/well to measure serum IgG against VSG3(S317A) carrier protein. Area under curve (AUC) after 45 min was calculated by GraphPad Prism. The circles, triangles and squares indicate sera at day −4, 58 and 68 respectively. Immunization with the conjugated trypanosome coats results in high IgG titers to fentanyl (average ˜150 μg/ml). Furthermore, the fact that soluble VSG3(S317A)-Fen (in PBS, without adjuvant) induced a secondary IgG response strongly indicates that immunization with Fen-conjugated VSG coats can induce a memory B cell response.
[0138] FIG. 10. Mice immunized with Fentanyl-haptenated T. brucei are protected from intoxication. A. Analgesic activity. Analgesic activity was tested by using the hotplate antinociception assay as described by Cox and Weinstock (1964). Fentanyl effect on hotplate antinociception was tested in unimmunized mice, in mice immunized with carrier only (VSG3(S317A)-only) or in mice immunized with haptenated VSG3(S317A)-Fen. In all cases mice were dosed with a cumulative fentanyl concentration of 0.1 mg/kg (s.c.). Fentanyl was administered subcutaneously every 15-30 minutes at increasing doses and the dose listed is the cumulative dose received. Hotplate antinociception was measured 15 minutes after the final fentanyl dose. Naloxone (0.1 mg/kg, s.c.) was administered 15 minutes after the final fentanyl dose. The effect of fentanyl is shown as latency to response. Fentanyl increased the latency to response after a cumulative dose of 0.1 mg/kg in unimmunized and VSG3-only immunized mice, compared with their baseline values. Naloxone completely reversed fentanyl-induced antinociception in both groups. Mice immunized with VSG3(S317A)-Fen did not show an increase in latency to response, compared to baseline, thus demonstrating that those mice did not get intoxicated by fentanyl at the same dose as the controls. Mean±standard deviation of 5 (unimmunized) or 6 mice per group are shown. B. Straub tail reaction (STR) measured per mouse per group. % denotes number of mice that demonstrated the Straub tail reaction, a dorsiflexion of the tail that is often almost vertical to the orientation of the body or curling back over the animal and stereotyped walking behavior (Bilbey et al, 1960). This phenomenon was first described as a response to opiates in mice (Straub, 1911), and is thought to be mediated by activation of the opioid receptor system because opioid receptor antagonists such as naloxone block the phenomenon (Aceto et al, 1969; Nath et al., 1994; Zarrindast et al, 2001).
[0139] FIG. 11. Each distinct VSG coat elicits a unique subset of B cell specificities (thus, a unique B cell repertoire). Trypanosomes expressing either VSG2 trypanosome coats or the VSG3(S317A) version used in the fentanyl vaccination experiments (a more immunogenic form of VSG3 with serine 317 mutated to alanine) elicit distinct repertoires in C57BL/6 mice. A. Gating strategy for the isolation of plasma cells. Lymphocytes were isolated from spleens and analyzed on a LSR II instrument for plasma cells. Plasma cells were then isolated (single cell sorted) using an ARIA II cell sorter. Cells were stained using an anti-mouse CD19-BV421 and anti-mouse CD138-BV510 antibodies. 7AAD was included in all stainings to exclude dead cells. The data were analyzed using FlowJo v10 software. Ig gene cloning was performed as described before (Tiller et al., 2008). In brief, cDNA of each single cell was generated using random hexamer primers. Ig heavy and corresponding Ig kappa or Ig lambda light chain gene transcripts were amplified using a semi-nested PCR strategy (Tiller et al., 2008). Amplicons were Sanger sequenced and analyzed using NCBI IgBlast. B. V gene repertoires elicited by VSG2 or VSG3(S317A) coated trypanosomes. Histograms summarize the VH and Vκ gene family usage in Ig gene transcripts isolated from plasma cells from C57BL/6 mice exposed to VSG2 or VSG3(S317A) coated trypanosomes (59 and 53 distinct Ig transcripts respectively). Sequences were obtained from 4 and 8 different VH gene families for VSG2 and VSG3(S317A), respectively, and 11 Vκ gene families. Within one bar the different shades of gray show the distribution of different family members within the respective family. The VH10 family, followed by the VH1 family (the largest VH family) was preferably expressed by the majority of plasma cells isolated from the VSG2 mice whereas only very few plasma cells expressing the VH10 family were isolated from the VSG3(S317A) mice. For VSG3 mice, most plasma cells expressed the VH1 family. In the VSG2 mice plasma cells mainly expressed the VK6 family, whereas in VSG3(S317A) mice the Vk family usage was more divers (but mainly VK1, VK4 and VK5). The analyzed sequences clearly demonstrate that the two different VSGs elicit a different VH and Vk family repertoire in plasma cells. This specificity allows one to design optimal vaccination strategies using distinct VSG coat platforms, tailored to the “needs” of specific epitopes. For example, this method can be used to expand an infrequently present B cell that is nevertheless required to be clonally expanded to produce an optimal response against a specific target.
[0140] FIG. 12: A. Illustrates the two types of particles that can be generated from trypanosomes. Immunization with large, intact, inactivated particles generates (FIG. 13) a traditional T-independent response. We have discovered that a subsequent boost with soluble VSG particles (i.e. the same material as the initial immunogen but now purified away from the coats in soluble form) is uniquely capable of generating a recall (memory) response together with very high antigen specific IgG titers. B. Shows vesicles derived from trypanosomes exhibiting a VSG coat.
[0141] FIG. 13: shows immunization with haptenated trypanosomes generates high titers of hapten-specific antibodies, and memory. A. Mice primed twice with the NP-haptenated entire VSG coat of trypanosomes followed by a boost with the same moiety (NP-haptenated entire VSG coat of trypanosomes) show a predominantly serum IgM response against NP and no IgG response. B. Mice primed twice with the NP-haptenated entire VSG coat of trypanosomes followed by a boost with the same moiety but now in soluble form show high serum IgG antibody titers again NP and memory. IgG titers are equivalent in proportion against both the carrier as well as the hapten. C. IgG titers elicited by our prime-boost combination are long lasting (e.g. still increasing even one month after the first boost, from Day 68 to Day 98). Data is shown for two different trypanosome strains expressing either VSG2 or VSG3. Data is presented as area under curve (AUC) from a serum dilution range from 1:100 to 1:12800. Each dot represents one mouse (n=5 mice per immunogen).
[0142] FIG. 14: Mice were primed and boosted with either 3E6, 1E7 or 5E7 NP-haptenated VSG3 coats subcutaneously. Mice receiving the highest dose of 5E7 NP-haptenated VSG3 coats showed the lowest NP specific serum IgG titers after the last bleed on Day 100. Thus dose escalation (increase) is not necessarily beneficial and potentially even detrimental to antigen-specific serum IgG titers.
[0143] FIG. 15: The specific prime-boost combination elicits high serum antibody titers against the hapten (here quantified for fentanyl haptenated to VSG3 which represent about 15% of total IgG in a mouse (2.38 mg/ml total IgG in a mouse) mouse (Klein-Schneegans et al., 1989)).
[0144] FIG. 16: Immunization with fentanyl-haptenated trypanosomes generates high titers of anti-Fen antibodies, and memory. A. Experimental timeline. B. ELISA titers presented as “area under curve” (AUC) for a dilution range from 1:100 to 1:12800. The ELISA plate is coated with Fentanyl-BSA (Fen-BSA). The immunogen (Fen-VSG3, VSG3 carrier alone or no immunogen) is noted on the x-axis). Each dot represents antiserum from 1 mouse (n=6 mice per immunogen) (4 time points per immunogen starting with pre-bleed—collected at day −4—up to post-2nd boost—collected at day 98—as per the timeline in A. C. ELISA titers presented as AUC for a dilution range from 1:100 to 1:12800 of antisera against the carrier. The ELISA plate is coated with VSG3. The immunogen (Fen-VSG3, VSG3 carrier alone or no immunogen) is noted on the x-axis.
[0145] FIG. 17: A comparison of vesicles to UV treated coats. Anti-fentanyl IgG normalized AUC is shown over a time course of 50 days. Prime boost on the left side was carried out with UV treated trypanosomal coats whereas vesicles were used on the right side. An apparently stronger increase in IgG levels was found for vesicles. However vesicles are more amenable to GMP production and have a longer shelf life.
[0146] The sequences show:
TABLE-US-00001 >Tb427VSG-1: GB Accession X56761.2 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.1 (Lister 427-1|Not fully assembled by me)| Source = GenBank download 170507| Protein length = 492 SEQ ID NO: 1 MATGRAKNTKWARWLSTAGLIIVVTLPATTMAAER TGLKATAWKPLCKLTTELSKVSGEMLNEGQEVISN IQKIKAAEYKVSIYLAKNPETQALQQLTLLRGYFA RKTNGGLESYKTMGLATQIRSARAAAYLKGSIDEF LNLLESLKGGSENKCLVTTNADTAATRRETKLDDQ ECALSMPETKPEAATRTELTQTGYPNLQHGGGGTA NTFQPTTSTGTCKLLSGHSTNGYPTTSALDTTAKV LAGYMTIPNTQVEATLANMQAMGNGHKATAPAWHE AWEARNREAKAKDLAYTNETGNLDTQPTLKALVKT LLLPKDNTEHNAEATKLEALFGGLAADKTKTYLDM VDAEIIPAGIAGRTTEAPLGKIHDTVELGDILSNY EMIAAQNVVTLKKNLDAVSKKQQTESAENKEKICN AAKDNQKACENLKEKGCVFNTESNKCELKKDVKEK LEKESKETEGKDEKANTTGSNSFLIHKAPLLLAFL LF >Tb427VSG-2: GB Accession X56762.1 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.2 (Lister 427-2|Identical in my assembly) | Source = GenBank download 170507 | Protein length = 476 SEQ ID NO: 2 MPSNQEARLFLAVLVLAQVLPILVDSAAEKGFKQA FWQPLCQVSEELDDQPKGALFTLQAAASKIQKMRD AALRASIYAEINHGTNRAKAAVIVANHYAMKADSG LEALKQTLSSQEVTATATASYLKGRIDEYLNLLLQ TKESGTSGCMMDTSGTNTVTKAGGTIGGVPCKLQL SPIQPKRPAATYLGKAGYVGLTRQADAANNFHDND AECRLASGHNTNGLGKSGQLSAAVTMAAGYVTVAN SQTAVTVQALDALQEASGAAHQPWIDAWKAKKALT GAETAEFRNETAGIAGKTGVTKLVEEALLKKKDSE ASEIQTELKKYFSGHENEQWTAIEKLISEQPVAQN LVGDNQPTKLGELEGNAKLTTILAYYRMETAGKFE VLTQKHKPAESQQQAAETEGSCNKKDQNECKSPCK WHNDAENKKCTLDKEEAKKVADETAKDGKTGNTNT TGSSNSFVISKTPLWLAVLLF >Tb427VSG-3: GB Accession AY935575.1 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.3 (Lister 427-3|Identical in my assembly) | Source = GenBank download 170507 | Protein length = 509 SEQ ID NO: 3 MQAAALLLLVLRAITSIEAAADDVNPDDNKEDFAV LCALAALANLQTTVPSIDTSGLAAYDNLQQLNLSL SSKEWKSLFNKAADSNGSPKQPPEGFQSDPTWRKQ WPIWVTAAAALKAENKEAAVLARAGLTNAPEELRN RARLALIPLLAQAEQIRDRLSEIQKQNEDTTPTAI AKALNKAVYGQDKETGAVYNSADCFSGNVADSTQN SCKAGNQASKATTVAATIVCVCHKKNGGNDAANAC GRLINHQSDAGANLATASSDFGDIIATCAARPPKP LTAAYLDSALAAVSARIRFKNGNGYLGKFKATGCT GSASEGLCVEYTALTAATMQNFYKIPWVKEISNVA EALKRTEKDAAESTLLSTWLKASENQGNSVAQKLI KVGDSKAVPPAQRQTQNKPGSNCNKNLKKSECKDS DGCKWNRTEETEGDFCKPKETGTENPAAGTGEGAA GANTETKKCSDKKTEGDCKDGCKWDGKECKDSSIL ATKKFALTVVSAAFVALLF >Tb427VSG-13: GB Accession AY935576.1 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.13 (Lister 427-13| Not fully assembled by me) | Source = GenBank download 170507 | Protein length = 499 SEQ ID NO: 4 MQRLGTAVFFLLAFRYSTEQAVGLKEPNAPCTTAC GCKSRLLKRLDLYTSKYADGINNERENSEAYSKLV TAALAAVPTMQRKILPLLGAAADILDICRRELATA RPLVQAAISKIEEAAGVYNTLHKLERGLGEAKIEF GGTDLRLTKTKFRATSLGTIHTADCPNADPGETNV KIGLEHEENEPEPAKLITHGHLDATCASGVGQSSS CHTTAVEANTHLTLGLTFSGSSKDESATWNAATNN KRAIHSNDADFLGSNATVAHEALKAIRSAGASTPC SSLITDFNAVRANPKFKLMVIKALLNKPTAEKESD APADEVNNAINSAYGREGSEYNTKTWKDIGSTRIP KADPPGEKTDTIDKLSSLPQWGDAIARLLLQEITK QEEQSIKTSSDEATNKECDKHTAKTEGECTKLGCD YDAENKKCKPKSEKETTAAGKKDRAAGETGCAKHG TDKDKCENDKSCKWENNACKDSSILATKKFALSMV SAAFVTLLF >X56767.1 | Trypanosoma brucei brucei | ILTat1 | mRNA variant surface protein ILTat 1.24 | Source = GenBank download 170421 | Protein length=514 SEQ ID NO: 5 MVYRNILQLSVLKVLLIVLIVEATHFGVKYELW QPECELTAELRKTAGVAKMKVN SDLNSFKTLELTKMKLLTFAAKFPESKEALTLRAL EAALNTDLRALRDNIANGIDRAVRATAYASEAAGA LFSGIQTLHDATDGTTYCLSASGQGSNGNAAMASQ GCKPLALPELLTEDSYNTDVISDKGFPKISPLTNA QGQGKSGECGLFQAASGAQATNTGVQFSGGSRINL GLGAIVASAAQQPTRPDLSDFSGTARNQADTLYGK AHASITELLQLAQGPKPGQTEVETMKLLAQKTAAL DSIKFQLAASTGKKTSDYKEDENLKTEYFGKTESN IEALWNKVKEEKVKGADPEDPSKESKISDLNTEEQ LQRVLDYYAVATMLKLAKQAEDIAKLETEIADQRG KSPEAECNKITEEPKCSEEKICSWHKEVKAGEKNC QFNSTKASKSGVPVTQTQTAGADTTAEKCKGKGEK DCKSPDCKWEGGTCKDSSILANKQFALSVASAAFV ALLF
EXAMPLES
[0147] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.
[0148] The examples show:
Example 1: Drug-Decorated VSG Coats
[0149] The inventors have generated tools to derivatize the dense and homogeneous surface coat of the African trypanosome (T. brucei) for use as a display platform for (any) antigens to which antibodies need to be raised. These tools consist of:
[0150] (a) A specific vector to efficiently replace the expressed VSG (VSG2) with any other VSG of interest (see below). FIG. 1 contains the maps of two such plasmid vectors.
[0151] (b) Specific sets of VSGs of interest: these contain extended N-termini that are accessible to the enzyme sortase (here, derived from Streptococcus pyogenes). N-termini accessibility is determined by (i) relative placement on the VSG (determined structurally—FIG. 2 contains VSG2 and VSG3 whose N-termini are accessible, and as a comparison also VSG13, whose N-terminus is not accessible because of steric hindrance). It is also determined by (ii) the initiating amino acids, which must be Ala-Ala for the particular sortase employed (or Gly-Gly for sortases from other organisms).
[0152] (c) VSGs that fulfil those criteria are engineered into a modified Lister 427 strain of T. brucei by replacing the active VSG (see (a) above—FIG. 4 contains the amino acid sequences of three such VSGs: VSG3 (A), VSG2 (B) and ILTat1.24 (C); It is worth mentioning that the VSG3 that is engineered for sortagging purposes, contains a mutation (S317A) that removes a native glycosylation event which the inventors have recently shown to be immune-suppressive—Pinger et al., Nat. Microbiology, 2018).
[0153] (d) The modification of the Lister 427 strain of T. brucei consists of genetic deletion of the endogenous glycophosphatidylinositol phospholipase C (GPI-PLC), the enzyme that “sheds” VSG off the surface of dying cells (this is crucial to generating T. brucei that can be used as vaccine display platform, because unless GPI-PLC is removed from the genome, any form of inactivation of the parasite (e.g. UV-irradiation), that is crucial (i) to disallow switching and loss of the engineered VSG and (ii) to remove infectivity, will also lead to the disintegration of the VSG coat and of the cell itself (once VSGs are shed due to the action of GPI-PLC, the VSG coat disintegrates and the cells lyse—FIG. 5 explains that concept).
[0154] The herein disclosed method depends on “highjacking” the natural ability of T. brucei to elicit a neutralizing (and long-lasting) antibody response to its VSG coat, to produce antibodies at will. The inventors do this by decorating the T. brucei VSG coat not only with any peptide epitope/antigen but also sugars, lipids or small molecules and then using the decorated VSG coat as a vaccine carrier. Specifically, the inventors use the enzyme sortase A to covalently ligate any moiety to VSG coats genetically engineered to carry N-terminal sortase acceptor sequences (FIGS. 4 and 6).
[0155] Therefore, the inventors produce a His-tagged sortase A, derived from Streptococcus pyogenes, in E. coli, using a plasmid containing the S. pyogenes-derived sortase A expression construct (pSpSortA-pET28a). This plasmid is transformed into BL21 DE3 cells (Life Technologies C6000-03). Colonies from this transformation are used to inoculate large cultures of LB media (Sigma-Aldrich, L3022-1KG) which are then grown shaking at 37° C. to an optical density (OD600) of 0.4-0.8. Cultures are induced with 1 mM IPTG, grown for an additional 3-4 h and harvested by centrifugation. Cell pellets are resuspended in TBS/imidazole (20 mM Tris, 150 mM NaCl, 20 mM imidazole), and lysed using an EmulsiFlex-05 homogenizer (Avestin). DNase-A powder (Sigma D5025) and 5 mM 2-Mercaptoethanol (2-ME) are added to the lysate, which is then clarified by centrifugation to remove particulates. The supernatant is passed through a column packed with Ni-NTA agarose beads (QIAGEN, 30230) equilibrated with Wash Buffer (20 mM Tris, 300 mM NaCl, 20 mM imidazole, 5 mM 2-ME). The column is then washed with 100 ml of Wash Buffer and eluted with 30-35 ml of Elution Buffer (20 mM Tris, 300 mM NaCl, 200 mM imidazole, 5 mM 2-ME). Samples containing protein are then pooled and dialyzed in Dialysis Buffer (20 mM Tris, 150 mM NaCl, 1 mM DTT). The resulting sample is concentrated using a centrifugal filter unit (Amicon Ultra-15, 10,000 NMWL, Merck Millipore), aliquoted and stored at −80° C. for future use.
[0156] The sortagging reaction is performed as follows: a mixture of sortagging solution containing 100 uM purified sortase A and 300-600 uM sortaggable-peptide in HMI-9 media is incubated on ice for 30-60 min (a sortaggable peptide includes any peptide with a C-terminal sortase donor sequence, LPSTGG, that can be attached at its N-terminus to another moiety; that moiety can be a fluorophore like 6-FAM, a small molecule like 4-Hydroxy-3-nitrophenyl acetyl (NP) hapten or other small molecules that are drugs of abuse (e.g. fentanyl etc.). GPI-PLC-negative T. brucei cells expressing engineered VSGs are then pelleted, resuspended in the sortagging mixture and incubated for 60 min at 4° C. on an inversion rotator. Cells are then pelleted, washed once with HMI-9 media and pelleted again before final resuspension in HMI-9 media (Hirumi and Hirumi, J. Parasitology, 1989). The efficiency of sortagging can be determined by direct FACS analysis or fluorescence microscopy (e.g. for fluorophores like 6-FAM) or by using specific monoclonal antibodies that bind the moieties decorating the VSG (FIG. 7 contains examples for 6-FAM, NP hapten and fentanyl hapten).
[0157] In proof of principle experiments this approach was used to generate (a) robust (in comparison to NP-CGG in Alum adjuvant) and (b) of consistent quality antibodies against a small-molecule hapten (4-hydroxy-3-nitrophenylacetyl or NP) (FIG. 8).
[0158] This approach can be used for a range of other small molecules (e.g. drugs of abuse like cocaine, nicotine, fentanyl, carfentanyl, tramadol, ketamine etc., but also chemotherapeutics like platinum, Adriamycin etc.; and also small molecules that are industrial by-products of chemical reactions), for toxins that mediate allergic reactions (e.g. aflatoxin and others) for specific peptides that function as important epitopes for infectious diseases (e.g. Plasmodium-derived peptides), for glycosylated or lipidated peptides (e.g. the aberrantly-glycosylated mucin peptides that have been considered as targets for anti-cancer vaccines etc.).
[0159] From the perspective of an anti-fentanyl (anti-overdose) vaccine, the major focus is to use this system to vaccinate “at risk” individuals (defined as individuals who are regular users or substance abusers but are not yet addicted/chemically dependent, or addicted individuals leaving rehabilitation centers, as proactive protection against overdose in case of recidivism, which typically will occur within the first two months after leaving rehab). Proof of concept that this has been achieved using the approach herein, is provided in FIGS. 9 and 10. Additionally, when this approach is coupled to repertoire analysis (FIG. 11), it will yield a wealth of anti-fentanyl monoclonal antibodies of varying affinities (directly accessible and ready to reconstitute from the paired immunoglobulin heavy and light chain sequences generated as a result of repertoire sequencing—FIG. 11). Such monoclonal antibody “sponges” can be used directly for therapeutic applications (e.g. anti-fentanyl antibody infusion together with methadone maintenance to curb bioavailability as well as cravings and accelerate therapeutic outcomes; or injection in the ER to blunt the effects of overdose in conjunction with naloxone—which acts quickly by antagonizing fentanyl binding to opioid receptors but which is metabolized faster than fentanyl, allowing delayed intoxication).
[0160] The Methodology: The ability of trypanosomes to stimulate a robust immune response in the infected individual (a response that is both long-lasting and neutralizing) is well documented. This invention renders this possible, at least in part, due to the discovery that a trypanosome's VSG protein is tolerant to the display of exogenous moieties with high efficiency on its surface using a bacterial transpeptidase sortase-based system (henceforth “sortagging”).
[0161] Specifically, a sortase acceptor sequence specific to the sortase (for sortase A derived from Streptococcus pyogenes that is Ala-Ala and for Sortase A derived from Staphylococcus aureus that is Gly-Gly) can be added at the exposed N-terminal part of the VSG protein which, when it gets transported to the surface of the trypanosome, remains accessible to the sortase (see FIGS. 2 and 6). It is noted that VSGs initiate with the Methionine of a signal peptide, but that peptide is cleaved upon maturation—hence the mature VSG sequence is not initiating with Methionine. For instance, both VSG2 and VSG3 (the preferred VSG variants) initiate with Ala-Ala, however the exact initiating amino acid must be empirically determined (using Edman degradation, which the inventors have done for both VSGs). Finally, while the endogenous Ala-Ala is present, it is inaccessible to sortase (and requires a short N-terminal extension as shown in FIG. 4). A complementary sortase donor sequence is then added C-terminally to the peptide/small molecule of interest (the actual sequence also depends on the sortase used; for sortase A derived from Streptococcus pyogenes, it is LPXTGG). LPXTGG can be added to a small molecule or other moiety with a reactive group (here the inventors use 6-FAM, or the hapten 4-hydroxy-3-nitrophenylacetyl abbreviated as NP, or Fen- FIG. 7).
[0162] For fentanyl, the N-terminus of the sortase sequence is linked via an amide bond to the fentanyl hapten at the position located in FIG. 7D. While most moieties can be derivatized, a derivatization that retains the antigenicity of the small molecule-peptide conjugate (e.g. NP-GGGSLPSTGG) must be empirically determined (for example this was done through trial and error for nicotine and other small molecules, e.g. NicVax). The sortase then ligates NP-carrying peptide to the exposed N-terminus of the VSG on the surface of the trypanosomes (the inventors validate this using anti-small molecule antibodies, FIG. 7). It is also possible to insert the sortase signal sequence within the loops of VSG (as done for FLAG peptide in Stavropoulos and Papavasiliou, 2010). However in such situations it is advisable to use the sortase donor sequence (e.g. LPXTGG) within the VSG loops and the sortase acceptor sequence (e.g. Ala-Ala) on the decorating small molecule, to increase specificity. Due to the dense coat of VSGs on the surface of trypanosomes, the small molecule is then densely displayed on the surface. Upon trypanosome injection into a mammalian host, the small molecule-conjugated VSG coat is exposed to the host immune system which mounts a similar strong and specific priming immune response against the exposed hapten (e.g. NP) as it does against the VSG in natural infection (FIG. 8). Boosting can then be achieved with hapten-VSG conjugate either on the full coat (FIG. 8) or formulations thereof (e.g. soluble haptenated VSG but other formulations as well) achieving a scalability that is unique to this vaccine platform.
[0163] Interestingly, primary responses elicited by different VSGs are not cross-reactive (i.e. antibodies raised to VSG2 do not cross-react with VSG3 etc. Pinger et al., Nat. Communications, 2017). This suggests that each specific VSG elicits a unique subset of B cell specificities (thus, a unique repertoire) which could be more or less potent toward a specific set of small-molecule haptens. In context of the invention, specific VSGs are selected for specific haptens, for the elicitation of optimal anti-hapten responses (with some VSGs better platforms for certain haptens—FIG. 7). For example, good anti-HA (pan-influenza) antibodies require engagement of IGHV1-69, a “rare” IGH within the general repertoire. A VSG that elicits IGHV1-69 might therefore be engaged preferably if one desired to use this method to elicit polyspecific anti-influenza antisera (e.g. in a “pan-flu” vaccine). Direct proof that VSGs elicit distinct B cell repertoires (and that this system can provide a range of different platforms depending on the preference of B cell to be elicited) is provided in FIG. 11.
[0164] Biosafety concerns (e.g. disease causation) but also a need to block natural switching away from the haptenated VSG, dictate that derivatized trypanosome coats are inactivated (and thus unable to cause infection). The inventors have achieved this via UV-crosslinking of trypanosomes that lack the enzyme glycophosphatidylinositol phospholipase C (GPI-PLC) and are therefore dead—but with an intact VSG coat (trypanosomes wildtype for GPI-PLC disintegrate upon UV-inactivation as GPI-PLC cleaves the GPI linkage of each VSG off the membrane and sheds the coat (as depicted in FIG. 5). UV-crosslinking (of GPI-PLC-negative trypanosomes is achieved by pelleting cells from culture, washing with irradiation buffer (PBS supplemented with 55 mM Glucose), and resuspending in the same buffer to a density of 10.sup.7 cells/ml. 1 ml of this suspension is then aliquoted into each well of a 6-well tissue culture plate (Thermoscientific, 150239). Plates are UV-irradiated for 8 cycles, each cycle 30 S using a UVP crosslinker (Analytik Jena). Plates are swirled between irradiation cycles to ensure equal irradiation of trypanosomes. Irradiated cells are then resuspended at a concentration of 15×10.sup.6 cells/ml. 200 μl of this solution (3×10.sup.6 trypanosomes) can be injected intraperitoneally or sub-cutaneously into mice.
[0165] Overall, using this inactivation protocol and sortaggable VSGs, the inventors have generated an optimal and flexible platform for the immunogenic display of antigenic determinants toward the generation of antibodies to small-molecule haptens and peptides, which can be expanded to a wide variety of antigenic entities (e.g. lipids, nucleic acids, etc.) Proof of concept regarding generation of antibodies to small molecules (e.g. NP) is shown in FIG. 9. Proof of principle that such antibodies can be raised against fentanyl and when generated protect against intoxication is provided in FIGS. 9 and 10. A cartoon version of the overall method is illustrated for the small molecule 6-FAM in FIG. 6.
[0166] An example of how to integrate the engineered VSGs of the invention into the T. brucei genome is provided in FIG. 3.
[0167] Generalizability of the approach: For the purposes of this application, the inventors focus on active immunotherapy against fentanyl, an adulterant of synthetic heroin and the cause of the majority of drug overdoses in the United States. This is because the inventors have tools already available (fentanyl haptenated to LPXTGG so that it can be sortagged; anti-fentanyl antibodies to verify sortaggability—from M. Pravetoni). However it should be clear that this approach can easily be adapted to raise effective antibodies against other drugs and drug metabolites (e.g. acetaminophen metabolites which cause liver toxicity, small molecules that are the toxins causal to anaphylactic shock in certain foodstuff allergies etc.). The approach can also be used for the haptenation with peptides derived from pathogens (e.g. the NANP tandem repeat, a major antigenic determinant of the Circumsporozoite protein of Plasmodium falciparum) or with aberrantly-glycosylated peptides unique to cancer cells (e.g. mucin) which can be used as anti-cancer vaccines (PMID: 20403708).
Example 2: A Unique Prime-Boost Combination for the Generation of High-Quality Antibody Responses and B Cell Memory
[0168] The immunization scheme developed in the experiments of FIGS. 8 and 9 above was further tested. The present invention is based on a different way to prime and boost, that does not require adjuvant but appears to elicit cognate T-cell help to small molecules in a physiological fashion. Specifically, the inventors used a hapten-carrier combination in a prime-boost combination (e.g. fentanyl covalently attached to trypanosome VSG protein) but in two completely distinct biophysical entities: the entire VSG coat of a trypanosome (a micrometer size particle which is highly dense and repetitive) followed by the same carrier moiety but now in soluble form (FIG. 12).
[0169] It was found that the haptenated VSG particle, despite its size, presents a highly immunodominant surface that can elicit a highly specific antibody repertoire and a predominantly IgM response (FIG. 13A). Conversely, it was found that the same moiety but now used as the soluble form during the boost, can elicit high IgG titers (equivalent in proportion to the carrier as to the hapten and also memory (FIG. 13B). Neither the priming nor the boost step requires adjuvant, suggesting that the invention is based on an, to date, unknown physiological mechanism to generate high antibody titers and memory. Furthermore, the amounts used are about 250× lower than those used in standard protocols (e.g. 100 μg NP-CGG per mouse), and lead to titers that are long lasting (e.g. still increasing even one month after the first boost) (FIG. 13C).
Example 3: Specific Priming Boosting Protocol—Dosing and Timing
[0170] Herein it was generally dosed twice within 30 days as the priming step (day 0 and day 30). This dual step is not essential but may help activate rare B-cell receptors from the germline repertoire. Then the inventors boosted at day 60 or day 60 and day 90. The day 60 boost leads to a recall IgG response that continues to increase in titers for the subsequent 30 days (FIG. 13C).
[0171] As mentioned, a dose that is about 250×lower than that delivered with standard methods is sufficient in the protocol of the invention. However, dose escalation (increase) at the priming step is not necessarily beneficial and potentially even detrimental (injection of material representing more than 50 million cells results in decreased titers, compared to injection representing 3 or 10 million cells—FIG. 14). An optimized protocol includes a priming dose representing about 5 million cells per bout 20 g of body weight—it resulted in an IgG response that is substantial (about 15% of total IgG in a mouse (2.38 mg/ml total IgG in a mouse (Klein-Schneegans et al., 1989))— FIG. 15). Thus, a two-step prime/boost process should be used in higher organisms as well, starting with a small dose as stated above.
[0172] In the below protocol details on the successful immunization schedule, using fentanyl as an example, are provided.
[0173] Six 6-8 weeks old female C57BL/6J mice per group were immunized with 5E6 cells of UV-irradiated sortagged VSG3-fentanyl cells (VSG3-Fen) subcutaneously (s.c.) in 200 μl of PBS at days 0 and 30. As control, 6 mice were either left unimmunized or injected with non-tagged UV-irradiated VSG3 carrier.
[0174] Two boosts were done using non-adjuvanted soluble VSG3 or VSG3-Fen in PBS at days 60 and 90. Blood samples were collected from the facial vein 4 days before starting the immunizations (day −4), 2 days before the first (day 58) and 8 days after the first (day 68) and second boosts (day 88 and day 98) with soluble VSG3 or VSG3-Fen proteins. [This material can be prepared ahead of time, concentrated and frozen in PBS; it can be thawed and diluted in PBS at the proper dose, prior to injection].
[0175] Results are shown in FIG. 16.
[0176] Specific IgG antibody titers against both Fen-hapten and VSG3 carrier protein in sera were measured by ELISA using 96-well plates coated with BSA-Fen conjugates or purified VSG3.
TABLE-US-00002 TABLE 1 Immunization Schedule: 1.sup.st Immunization 2.sup.nd Immunization 3.sup.rd Immunization 4.sup.th Immunization Group (Day 0, S.C.) (Day 30, S.C.) (Day 60, S.C.) (Day 90, S.C.) VSG3-Fen VSG3-Fen cells VSG3-Fen cells 100 μg VSG3-Fen in 100 μg VSG3-Fen in (5E6 cells, in PBS (5E6 cells, in PBS PBS (no adjuvant) PBS (no adjuvant) or PBS-glucose) or PBS-glucose) VSG3 (carrier VSG3 cells (5E6 VSG3 cells (5E6 100 μg VSG3 in 100 μg VSG3 in control) cells, in PBS) cells, in PBS) PBS (no adjuvant) PBS (no adjuvant) non-immunized
Example 4: Comparing Vesicles to UV Treated Coats
[0177] In order to compare long term efficacy of vaccination using vesicles to UV treated coats, immunization was carried out as described in Example 3 using vesicles containing VSG3-Fen to UV treated trypanosomal coats.
[0178] Anti-fentanyl IgG titers (normalized AUC values) were determined over a time course comprising day 2, 12, 22, 40 and 50 measurements; see FIG. 17, priming on the left side was carried out with UV treated trypanosomal coats whereas vesicles were used on the right side. Boosting was carried out in both groups using soluble VSG as described before. A significantly stronger increase in IgG titers was found for vesicles.
[0179] Accordingly, vaccination using vesicles is apparently superior over vaccination using UV treated coats. Moreover, vesicles are more amenable to GMP production and have a longer shelf life.
