Immunization scheme for mutant surface glycoprotein carriers

Abstract

The present invention relates 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 array-displayed VSG variants and soluble VSG variants. Surprisingly, the immunization scheme of the present invention elicits strong and long-lasting antibody responses in immunized subjects and is therefore applicable to vaccination approaches, immunotherapy, and the generation of novel antibodies.

Description

[Technical field]

[0001] The present invention relates 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 array-displayed and soluble VSG variants. Surprisingly, the immunization scheme of the present invention induces a strong and long-lasting antibody response in the immunized subject and is therefore applicable to vaccination approaches, immunotherapy and the generation of novel antibodies. [Background technology]

[0002] Drug abuse and addiction to drugs pose significant problems for the medical, social and economic well-being of both individuals and their families, and society as a whole. Particularly in the United States, abuse of prescription opioid painkillers, such as OxyContin® and Vicodin®, has reached epidemic levels and is a public health crisis of current grave concern. In other regions (e.g., Asia, Africa and the Middle East), tramadol abuse is on the rise (UNODC World Drug Report 2017). All aspects of society are affected by the relative availability, perceived safety and overprescription of these drugs by medical prescribers.

[0003] The pathway to addiction often stems from medically authorized administration for pain management, but for a significant proportion of the population (estimated 10-15%), this can rapidly lead to addiction and then illicit use of prescription opioids. Of particular concern is the widespread illicit use of these drugs by teens and the sharp increase in emergency room visits (reaching 1.2 million in 2009, DAWN report, 2010) as well as the rapid increase in cases of fatal overdoses due to abuse (overdose deaths were estimated at 72,000 in 2017, surpassing breast cancer deaths for the first time, with no end in sight). Currently, treatment options are limited to supervised, mediated, and controlled withdrawal in rehab / detoxification clinics (e.g., with methadone or buprenorphine).

[0004] Methadone maintenance therapy, developed at Rockefeller University for the treatment of heroin addiction, remains the most effective and widely used pharmacological treatment for any addictive disorder (Dole et al., 1966; Kreek, 2000). However, treatment often requires several years and retention rates are very low, which has led to increased interest in additional options and / or preventative drugs for abuse in the first place. One emerging approach to addiction is vaccines that generate neutralizing antibodies against specific drug targets (Shen et al., 2012), an option that has been explored most extensively for cocaine (Fox et al., 1996; Shen and Kosten, 2011) and more recently for heroin derivatives (Pravetoni et al., 2012, 2017 and 2018). The idea of ​​immunotherapy for treating opioid use disorder (or at least preventing overdose) is based on the concept that if a drug conjugated to a carrier protein could induce a long-lasting immune response, antibodies against the drug's chemical structure would develop, and the circulating antibodies would then bind to the drug upon administration by the user and prevent it from reaching its site of action in the brain.

[0005] Successful vaccination is defined as high antibody titers and induction of memory B cells. High titers arise from the recruitment of antigen-specific B cells, their activation and differentiation into antibody-producing plasmablasts. Memory B cells can further induce recall (memory) responses that require cognate T cell help. Most successful vaccines consist of a single component (e.g., virus particles) in an adjuvant, given as a priming immunization and multiple subsequent boosts. However, this is not effective for vaccines against small molecules or isolated short, well-defined epitopes. The immune system responds physiologically to large (nanometer to micrometer) entities (viruses, bacteria), but not to entities the size of a few nucleotides or amino acids. In the context of antibody induction against small molecules or defined small epitopes in general, recruitment of specific B cells can only be induced (meaning to "artificially" induce cognate T cell help) if the small epitope (also known as a hapten) is bound to a larger protein (known as a carrier) and delivered with an adjuvant. However, because the hapten is a small fraction of the size of the carrier, most of the immune response occurs against the carrier, including the generation of cognate T cell help. As a result, this approach can generate reasonable titers, but has failed to generate memory every time it has been attempted. The use of antigenic particles, such as inactivated trypanosomes, has also been described for VSG-based vaccination using haptens (WO2020 / 084072). Summary of the Invention [Problem to be solved by the invention]

[0006] It is therefore an object of the present invention to provide a vaccination and immunization approach that allows the generation of a stable and long-lasting memory immune response in a subject. [Means for solving the problem]

[0007] Brief description of the invention In general, and for purposes of brief description, the main aspects of the invention can be described as follows.

[0008] In a first aspect, the present invention relates to a method for inducing an immune response and / or amplifying an 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, wherein (i) the at least one priming immunization step comprises immunization of the subject with an array of immunization carrier fusions on vesicles or beads, each immunization carrier fusion consisting of a carrier protein fused to an antigenic compound, and (ii) the at least one boosting immunization step comprises immunization of the subject with the immunization carrier fusions used in (i) in a non-array form, preferably in a soluble form.

[0009] In a second aspect, the present invention relates to a method for producing antibodies against an antigenic compound, comprising the steps of performing the method of the first aspect in a non-human animal and isolating from the non-human animal newly produced antibodies or antibody-producing B cells, wherein the antibodies are against the antigenic compound.

[0010] In a third aspect, the present invention relates to an antibody or an antibody-producing B cell generated and / or isolated according to the method of the second aspect.

[0011] In a fourth aspect, the present invention relates to an immunization kit comprising (a) an array or aggregate of immunization carrier proteins on vesicles or beads as described in the first aspect, and (b) a non-aggregated and / or non-arrayed form, preferably a soluble form, of the immunization carrier proteins of (a), wherein the immunization carrier proteins of (a) and (b) can be fused with an antigenic compound to obtain the immunization carrier fusion as described in the first aspect.

[0012] In a fifth aspect, the present invention relates to a method for treating or preventing an adverse condition in a subject by inducing an immune response and / or amplifying an existing immune response to an antigenic compound causing or associated with the adverse condition, comprising carrying out the steps of the method of the first aspect.

[0013] The drawings show: [Brief description of the drawings]

[0014] [Figure 1A] The maps of the plasmids used for VSG modification are shown. The sequences of pHH-VSG3.G4S-Hyg and pHH-ILTat1.24-G4S-Hyg plasmids are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively. (A) pHH-VSG3.G4S-Hyg plasmid size: 7204 bp. VSG2-CTR (VSG2 cotransfer 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 sequence: 4715-4915, Ori (Bacterial replication origin): 5385-5973, AmpR (Ampicillin resistance gene, beta-lactamase): 6144-7004 (reverse complement), AmpR promoter (Ampicillin resistance gene promoter, beta-lactamase promoter): 7005-7109 (reverse complement). [Figure 1B]The maps of the plasmids used for VSG modification are shown. The sequences of pHH-VSG3.G4S-Hyg and pHH-ILTat1.24-G4S-Hyg plasmids are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively. (B) pHH-ILTat1.24-G4S-Hyg, plasmid size: 7219 bp. VSG2-CTR (VSG2 cotransfer 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 sequence: 4730-4930, Ori (Bacterial replication origin): 5400-5988, AmpR (Ampicillin resistance gene, beta-lactamase): 6159-7019 (reverse complement), AmpR promoter (Ampicillin resistance gene promoter, beta-lactamase promoter): 7020-7124 (reverse complement). [Figure 1C]Maps of the plasmids used for VSG modification are shown. The sequences of pHH-VSG3.G4S-Hyg and pHH-ILTat1.24-G4S-Hyg plasmids are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively. (C) Importantly the sequence of pPNSM2 encoding "sortagable VSG3" (synonymous with "VSG3-G4S(S317A)") is provided in SEQ ID NO:6. pPNSM2 plasmid size: 7060 bp. Important restriction enzyme sites for cloning and cell line generation are marked in bold, offering two options for linearization prior to transfection. Important elements of the plasmid are also marked in order at the following positions: BES1 cotransfer region (1-1227, acting as a homology region facilitating genomic integration), sortagging-capable VSG3 (1247-2821), VSG 3UTR (2828-2896), actin 5'-UTR (3087-3193), Hygro (3219-4244, representing the open reading frame of the hygromycin resistance gene), aldolase 3'-UTR (4255-4395), ori (5020-5608, representing the bacterial origin of replication on the bottom strand), AMP_R (5779-6639, representing the bacterial ampicillin resistance cassette on the bottom strand), and telomere seed (6785-6985). Plasmids for VSG2 and ILtat1.2 can be made based on the plasmid described for VSG3 above by replacing the solderable VSG3 with VSG2 or ILtat1.2. [Diagram 2]Crystallographic studies to help determine the accessibility of VSG N-termini to sortase enzymes are shown with five examples (all published except VSG13). "N" represents the position of the free N-terminus for each protein. The lines represent the roughly estimated general positions of surface-exposed residues (the "top" of the VSG coat accessible to sortase enzymes). Many VSGs can be engineered to accept tags via sortase-based conjugation (we have already done this for VSG2, VSG3 and ILTat1.24, as described below), but not all do (for example, the N-terminus of VSG13 is largely buried and may not be fully accessible). Structural biology has demonstrated very useful pre-screening of VSGs, revealing many surface elements and other structural features that inform VSG selection (for example, the discovery of O-linked sugars on VSG3 led us to remove it from the surface with the mutation S317A, which turns a specific serine into an alanine). [Figure 3-1] Figure 1 shows the strategy for knocking-in sortagable VSGs into the genome of Trypanosoma brucei. A to C show the genetic cloning strategy for expressing modified VSG2, VSG3 and ILTat1.24 from active expression sites of endogenous VSGs (replacing wild-type VSG2). [Figure 3-2] Continued from Figure 3-1. [Figure 4-1]The amino acid sequences of the sortagable VSG proteins are shown. A. Amino acid sequence of the sortagable VSG3.G4S(S317A) protein. The signal peptide is underlined. The mature sortagable VSG3 is derived from a more antigenic variant (S317A) of wild-type VSG3. VSG3-G4S starts with a dialanine (bold and italic). This dipeptide is the acceptor for the sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The N-terminal extension (by addition of the (G4S)3 peptide linker, in bold) is important for sortase A's ability to access the dialanine. B. Amino acid sequence of the sortagable VSG2 protein (VSG2-1DK). The signal peptide is underlined. The mature sortagable VSG2-1DK starts with a dialanine (bold and italic). This dipeptide serves as an acceptor for the sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The N-terminal extension (due to the addition of a linker peptide consisting of a TEV protease cleavage site flanked by polyglycines, in bold) is important for sortase A's ability to access the di-alanine. C. Amino acid sequence of the sortagable ILTat1.24 protein (ILTat1.24-G4S). The signal peptide is underlined. The mature sortagable ILTat1.24-G4S starts with a tetraglycine (bold and italics). This tetraglycine serves as an acceptor for the sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The VSG N-terminal extension (due to the addition of a (G4S)2 peptide linker, in bold) is important for sortase A's ability to access the tetraglycine. [Figure 4-2] Continued from Figure 4-1. [Diagram 5]Wild-type Trypanosoma brucei sheds its VSG coat upon death. A. Schematic of the process showing that GPI-PLC is activated after cell death. B. The location of the GPI-anchor cleavage site is indicated. Cleavage of the GPI anchor releases (sheds) the VSG from the coat and the uncoated trypanosome disintegrates due to osmotic pressure (and consequently loses its immunogenic properties). [Figure 6] Schematic of the overall method for sortagging the VSG coat of trypanosomes. A. Visualization of a VSG protein homodimer embedded in the trypanosome membrane via a glycophosphatidylinositol (GPI) anchor. Bottom: Whole trypanosome. Top: Zoomed-in view of one VSG protein homodimer. B. Schematic of the sortagging reaction: A modified VSG protein (with an N-terminal dialanine) and a small molecule (oval) linked to the sortase donor sequence LPSTGG are covalently attached via a sortase reaction. [Figure 7-1] Methods for detecting sortagging efficiency are shown. A. Top image: sortagging of VSG2-1DK (left) and VSG3-G4S(S317A) (right) detected by direct fluorescence (6-FAM). Fluorescence microscopy images of Trypanosoma brucei cells are shown on top (left: sortagged VSG2-1DK, right: sortagged VSG3-G4S). Bottom image: 6-FAM sortagged VSGs were also analyzed by flow cytometric analysis using a FACSCalibur. B. Sortagging of VSG2-1DK and VSG3-G4S(S317A) detected by FACS analysis of trypanosomes using a monoclonal antibody against a small molecular moiety (4-hydroxy-3-nitrophenylacetyl, abbreviated here as NP) followed by staining with an allophycocyanin (APC)-conjugated mouse monoclonal IgG antibody (B1-8 clone, Abcam). [Figure 7-2]A method for detecting sortagging efficiency is shown. C. 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 the fentanyl conjugate compound is alternatively designated as "Fen-" or "-Fen"). The peptide bearing the fentanyl hapten was conjugated to three different genetically modified VSGs (VSG2, VSG3, and ILTat1.24) using sortase A as described above. The chemical synthesis process of fentanyl hapten is described in MD Raleigh et al., J. Pharmacol. Exp. Ther. 368:282-291, 2019, and is adapted here for sortase-mediated conjugation. D. After sortase A-mediated conjugation of fentanyl hapten to VSG, 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 VSG, followed by flow cytometric analysis using a FACS-Calibur. Non-tagged VSG was used as a control for background staining. The mode and median of the data set are shown at the bottom of the graph. In both fentanyl and FAM conjugation, ILTat1.24 was superior to VSG2 and VSG3. Also, the sortagging efficiency of VSG3 was moderately higher than that of VSG2. [Figure 8-1]Comparison of antibody responses to the small molecule 4-hydroxy-3-nitrophenylacetyl (NP): immunization with NP-labeled Trypanosoma brucei versus the "gold standard" hapten-carrier conjugate (i.e., NP-conjugated chicken gamma-globulin (NP-CGG) in Alum adjuvant). Five 6- to 8-week-old female C57BL / 6J mice per group were primed on days 0, 3, and 30 with intact VSG3(S317A) or VSG3-NP-coated (i.e., UV-irradiated intact Trypanosoma brucei cells expressing sortagable VSG3(S317A) tagged or not with the NP hapten, no adjuvant) or NP-CGG in Alum adjuvant. On day 70, the mice were boosted with soluble VSG3(S317A)-NP (in PBS, no adjuvant) (or boosted with soluble NP-CGG in PBS without adjuvant for the control group). A. Priming immunization with VSG-NP on intact trypanosomes and subsequent boosting with soluble VSG3(S317A)-NP shows clear IgG recall responses and leads to the generation of substantial IgG titers against the small molecule hapten NP. Titers were measured before and after boost and are shown as serum dilution 1:800. 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 conjugated trypanosome coat leads to high IgG titers against NP (average approx. 500 μg / ml). Furthermore, soluble VSG3-NP (in PBS, no adjuvant) induced a strong secondary IgG response, indicating that immunization with NP-conjugated VSG coats can induce memory B cell responses. Although the average titers generated against NP-CGG in Alum were slightly higher, boosting with soluble NP-CGG (in PBS, no adjuvant) did not induce robust secondary responses, indicating the absence of memory B cell responses after priming with NP-CGG in Alum.B. Immunization with VSG-NP on intact trypanosome coats and subsequent boosting with soluble VSG3(S317A)NP results in high affinity IgG (defined by the NP2 / NP30 IgG titer ratio). The increase in the NP2-BSA / NP30-BSA IgG ratio in the VSG3-NP group indicates affinity maturation, characteristic of memory B cell (recall) responses. Immunization with the "gold standard" hapten-carrier conjugate in Alum (NP-CGG) does not result in any increase in the affinity of anti-NP IgG antibodies. C. Immunization with VSG-NP on intact trypanosome coats and subsequent boosting with soluble VSG3(S317A)-NP also produces anti-VSG3 (anti-carrier) antibodies, which are of a similar magnitude (μg / ml) as antisera produced against the NP hapten (quantified by serial dilutions of anti-VSG3 mouse monoclonal IgG (11D6 clone)). Further data show no immunological cross-reactivity between VSG2 and the VSG3 carrier (not shown). [Figure 8-2] Continued from Figure 8-1. [Figure 9-1]Antibody responses to fentanyl induced by Fen-labeled Trypanosoma brucei are shown to achieve high titers and memory (recall). ELISA measurements of serum IgG against the fentanyl hapten and VSG3 carrier protein are shown. A tetraglycine peptide with an AN-terminal fentanyl hapten (Fen-G4) was synthesized as described above. 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- to 8-week-old female C57BL / 6J mice per group were primed by subcutaneous injection with intact VSG3(S317A) or VSG3(S317A)-Fent coated (i.e., UV-irradiated intact Trypanosoma brucei cells expressing sortagable VSG3(S317A) tagged or untagged with Fen-hapten, no adjuvant) on days 0 and 30. The mice were then given a soluble VSG3(S317A)-Fent (in PBS, no adjuvant) booster on day 60. Serum samples tested included pre-immunization (day -4), 2 days before (day 58) and 8 days after (day 68) one boost with soluble VSG3(S317A)-fentanyl protein. Anti-fentanyl IgG in serum was detected using anti-mouse HRP conjugate (1:3000) followed by the addition of ABTS substrate and H2O2 prepared in citrate phosphate buffer pH 4.2. The absorbance of the samples at A405 nm was measured after 45 min using an ELISA reader (Tecan, Infinite M1000 Pro). B. To quantify the IgG concentration in serum samples, a standard curve was generated using serial dilutions of mouse monoclonal antibodies against Fen-hapten. The mean ± standard deviation of six mice per group is shown. C. Similarly, 96-well plates were coated with FPLC-purified VSG3(S317A) protein at 5 μg / well and serum IgG against VSG3(S317A) carrier protein was measured. The area under the curve (AUC) after 45 min was calculated using GraphPad Prism. Circles, triangles and squares represent sera at days -4, 58 and 68, respectively.Immunization with conjugated Trypanosome coats results in high IgG titers to fentanyl (average ∼150 μg / ml). Furthermore, soluble VSG3(S317A)-Fen (in PBS, no adjuvant) strongly induced secondary IgG responses, indicating that immunization with Fen-conjugated VSG coats can induce memory B cell responses. [Figure 9-2] Continued from Figure 9-1. [Figure 10]We show that mice immunized with fentanyl-haptenized Trypanosoma brucei are protected from intoxication. A. Analgesic Activity. Analgesic activity was tested using the hot plate antinociception assay as described by Cox and Weinstock (1964). Fentanyl effects on hot plate antinociception were tested in non-immunized mice, mice immunized with carrier only (VSG3(S317A) only), or mice immunized with haptenized VSG3(S317A)-Fen. In all cases, mice were administered a cumulative fentanyl concentration of 0.1 mg / kg (sc). Ascending doses of fentanyl were administered subcutaneously every 15 to 30 minutes, and the doses listed are the cumulative doses administered. Hot plate antinociception was measured 15 minutes after the last fentanyl administration. Naloxone (0.1 mg / kg, sc) was administered 15 minutes after the last fentanyl administration. The effect of fentanyl is shown as the latency to respond. Fentanyl increased the latency to respond after a cumulative dose of 0.1 mg / kg compared to baseline values ​​in non-immunized mice and mice immunized with VSG3 alone. Naloxone completely reversed fentanyl-induced antinociception in both groups. Mice immunized with VSG3(S317A)-Fen showed no increase in the latency to respond compared to baseline, thus indicating that these mice were not addicted to the same dose of fentanyl as the controls. The mean ± standard deviation of 5 (non-immunized) or 6 mice per group is shown. B. Straub tail response (STR) measured per mouse per group. The % represents the number of mice that showed Straub tail response, dorsiflexion of the tail, often nearly perpendicular to the direction of the body or curled up above 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). [Figure 11-1]We show that each different VSG coat induces a unique subset of B cell specificities (and therefore a unique B cell repertoire). Trypanosomes expressing either the VSG2 trypanosome coat used in the fentanyl vaccination study or the VSG3(S317A) version (a more immunogenic form of VSG3 with serine 317 mutated to alanine) induce different repertoires in C57BL / 6 mice. A. Gating strategy for isolation of plasma cells. Lymphocytes were isolated from the spleen and analyzed on an LSR II instrument for plasma cells. Plasma cells were then isolated using an ARIA II cell sorter (single cell sorting). Cells were stained with anti-mouse CD19-BV421 and anti-mouse CD138-BV510 antibodies. 7AAD was included in all stainings to exclude dead cells. Data were analyzed using FlowJo v10 software. Ig gene cloning was performed as described previously (Tiller et al., 2008). Briefly, cDNA of each single cell was generated using random hexamer primers. Ig heavy chain 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 induced by VSG2 or VSG3(S317A)-coated trypanosomes. The histograms outline 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 distinct VH gene families and 11 Vκ gene families for VSG2 and VSG3(S317A), respectively. Within a bar, different grey shades indicate the distribution of different family members within each family. The VH10 family, followed by the VH1 family (the largest VH family), was preferentially expressed by the majority of plasma cells isolated from VSG2 mice, whereas only a few plasma cells expressing the VH10 family were isolated from VSG3(S317A) mice.For VSG3 mice, most plasma cells expressed the VH1 family. In VSG2 mice, plasma cells expressed mainly the VK6 family, whereas in VSG3(S317A) mice, Vk family usage was more diverse (mainly VK1, VK4 and VK5). The analyzed sequences clearly show that the two different VSGs induce different VH and Vk family repertoires in plasma cells. This specificity allows optimal vaccination strategies to be designed using different VSG coating platforms tailored to specific epitope "needs". For example, this method can be used to expand B cells that are rare but nevertheless need to be clonally expanded to provide optimal responses against specific targets. [Figure 11-2] Continued from Figure 11-1. [Figure 12] A. Shows two types of particles that can be made from Trypanosoma. Immunization with large intact inactivated particles generates a traditional T-independent response (Figure 13). We have discovered that a subsequent boost with soluble VSG particles (i.e., the same material as the initial immunogen, but now purified separately from the coat in soluble form) can intrinsically generate a recall (memory) response, along with very high antigen-specific IgG titers. B. Shows vesicles derived from Trypanosoma showing the VSG coat. [Figure 13A] We show that immunization with haptenized trypanosomes generates high titers of hapten-specific antibodies and memory. Mice primed twice with A. brucei NP-haptenized whole VSG coat and then boosted with the same moiety (A. brucei NP-haptenized whole VSG coat) show primarily serum IgM and no IgG responses to NP. [Figure 13B]We show that immunization with haptenized trypanosomes produces high titers of hapten-specific antibodies and memory. B. Mice primed twice with NP-haptenized whole VSG coat of Trypanosoma brucei and then boosted with the same moiety, but now in soluble form, show high serum IgG antibody titers and memory against NP. IgG titers are proportionally equivalent against both the carrier and the hapten. [Figure 13C] We show that immunization with haptenized trypanosomes generates high titers of hapten-specific antibodies and memory. C. IgG titers induced by our prime-boost combination are long-lasting (e.g., increasing from day 68 to day 98, one month after the first boost). Data are shown for two different trypanosome lineages expressing either VSG2 or VSG3. Data are presented as area under the curve (AUC) from a serum dilution range of 1:100 to 1:12800. Each dot represents one mouse (n=5 mice per immunogen). [Figure 14] Mice were subcutaneously primed and boosted with either 3E6, 1E7 or 5E7 NP-haptenylated VSG3 coats. Mice receiving the highest dose of 5E7 NP-haptenylated VSG3 coats showed the lowest NP-specific serum IgG titers after the last bleed on day 100. Thus, dose escalation is not necessarily beneficial and may even be potentially detrimental to antigen-specific serum IgG titers. [Figure 15] The specific prime-boost combination induces high serum antibody titers to the hapten (here quantified for fentanyl haptenized to VSG3, which represents approximately 15% of total IgG in mice (2.38 mg / ml total IgG in mice (Klein-Schneegans et al., 1989)). [Figure 16-1]Immunization with fentanyl-haptenized trypanosomes generates high titers of anti-Fen antibodies and memory. A. Experimental timeline. B. ELISA titers expressed as "area under the curve" (AUC) for a dilution range of 1:100 to 1:12800. ELISA plates are coated with fentanyl-BSA (Fen-BSA). Immunogen (Fen-VSG3, VSG3 carrier alone, or no immunogen) is indicated on the x-axis. Each dot represents antiserum from one mouse (n=6 per immunogen) (following the timeline in A, starting with a pre-bleed on day -4 and ending with a post-2 boost bleed on day 98, 4 time points per immunogen). [Figure 16-2] Immunization with fentanyl-haptenized trypanosomes produces high titers of anti-Fen antibodies and memory. C. ELISA titers expressed as AUC for a dilution range of 1:100 to 1:12800 of antisera against carrier. ELISA plates are coated with VSG3. Immunogen (Fen-VSG3, VSG3 carrier alone, or no immunogen) is indicated on the x-axis. [Figure 17] Comparison of vesicles and UV-treated coat. AUC normalized to anti-fentanyl IgG is shown over a 50-day time course. Prime-boost on the left was performed with UV-treated trypanosome coat, while on the right vesicles were used. A clearly stronger increase in IgG levels was found for vesicles. However, vesicles are more suitable for GMP manufacturing and have a longer shelf life. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The elements of the present invention are described below. Although these elements are described with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This specification should be understood to support and encompass embodiments combining two or more of the explicitly described embodiments, or combining one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described herein should be considered to be disclosed herein, unless the context indicates otherwise.

[0016] Definition: As used herein, the terms "of the invention," "according to the invention," and "according to the present invention" and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0017] The term "comprising" as used herein is to be interpreted as embracing both "including" and "consisting of," both meanings being specifically intended and therefore individually disclosed embodiments according to the invention. As used herein, "and / or" should be interpreted as specifically describing each of two specific features or components, with or without the other. For example, "A and / or B" should be interpreted as specifically describing each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein. In the context of the present invention, the terms "about" and "approximately" represent an interval of precision where a person skilled in the art would understand that the technical effect of the feature in question is still certain. The terms typically indicate a deviation of ±20%, ±15%, ±10%, e.g. ±5% from the indicated numerical value. As a person skilled in the art would understand, such a specific deviation for a numerical value relating to a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than man-made or engineered technical effects. As will be understood by one of ordinary skill in the art, the specific such deviations for the numerical values ​​for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than man-made or engineered technical effects. When an indefinite or definite article is used when referring to a singular noun, such as "a," "an," or "the," this includes the plural of that noun unless otherwise specified.

[0018] The term "antigenicity" in the context of the present invention shall refer to the characteristic of the particles of the invention to induce a specific immune response when used as an immunogen in a vaccination or immunization procedure.

[0019] The term "variant surface glycoprotein", abbreviated as "VSG", refers to a family of approximately 60 kDa proteins clustered on the cell surface of protozoan parasites belonging to the genus Trypanosoma. The parasite has a rich cellular repertoire of antigenically distinct VSGs (approximately 1500 complete and partial (pseudogenes)) that are expressed from bloodstream expression sites (BES, ES) together with other ES-related genes (ESAGs) (of which the transferrin receptor (Tfr: ESAG6, ESAG7) is one) in a polycistronic manner by RNA polymerase I (recruited to a ribosomal promoter). Only one VSG gene is expressed at a time, since only one of the approximately 15 ES is active in the cell. VSG expression is "switched" by homologous recombination induced by the transfer of silent basic copy genes from the array to active telomerically-located expression sites (homology-induced) through double-strand breaks. VSG annotations, protein sequences and gene sequences are available from public databases, e.g. www.ensemble.org. A VSG collection for Trypanosoma brucei brucei (Lister 427 strain) is published in Cross GA et al., Mol. Biochem. Parasitol. 2014 Jun; 195(1):59-73. Doi:10.1016 / j.molbiopara.2014.06.004. A preferred VSG in the context of the present invention is Trypanosoma brucei VSG, more preferably VSG1, VSG2, VSG3 or ILTat1.24.The VSG according to the invention is preferably characterized by an N-terminus three-dimensionally located within the VSG in a position that is sufficiently close to the outer coat surface to be accessible if the VSG is present, for example, within the VSG coat on a trypanosome cell, and the addition of a linker sequence (preferably 100 amino acids or less, preferably 50 amino acids or less, most preferably 20 amino acids or less in length) allows modification of the N-terminus of the VSG protein with or without such extension. Any sequence inserted N-terminally into the VSG sequence is preferably inserted immediately before the initiating methionine (M). In a further embodiment, the insertion is immediately downstream of the signal peptide cleavage site. A preferred VSG sequence for use in the context of the present invention is a VSG comprising any one of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0020] A "VSG type" or "type of VSG" refers to a family member of the VSG family, preferably one of the trypanosoma VSGs.

[0021] The term "engineered VSG" or "eVSG" refers to any VSG protein that contains an artificial modification compared to the wild-type sequence of said VSG. Preferably, the eVSG is a non-naturally occurring sequence. A VSG may be engineered to be an eVSG by either post-translational modification of the VSG coding sequence, chemical modification, genetic engineering, and any other method known to those skilled in the art for protein modification.

[0022] The term "immunogenic compound" or "immunogen" as used herein encompasses any type of compound or structure capable of eliciting an immune response in a host. Immunogenic compounds according to the present invention preferably, but not necessarily, comprise an amino acid sequence or are small molecule compounds. A small molecule in the context of the invention described herein is any compound that has a molecular mass significantly lower than, for example, complex macromolecules or proteins, and thus preferably has a molecular weight of less than 50 kDa, preferably less than 20 or 10 kDa.

[0023] The term "immunization carrier fusion" in the context of the present invention is a fusion of a carrier protein suitable for immunization purposes, preferably covalently or non-covalently fused to an immunogen or antigenic compound against which an antibody or immune response is generated according to the present invention. The carrier protein is preferably VSG or a variant thereof as described herein. The immunogen or antigen is also as described herein, but is preferably a small molecule, e.g. a small molecule drug.

[0024] An "array" or "aggregate" of immunized-carrier fusions according to the invention disclosed herein relates to an ordered arrangement of a plurality of such proteins in close proximity to one another. An array or aggregate of carrier proteins or immunized-carrier fusions is provided on a vesicle or bead according to the invention.

[0025] In contrast, the term "soluble immunization-carrier fusion" refers to a single immunization-carrier fusion protein that has been solubilized.

[0026] The term "sortase" as used herein refers to a protein with sortase activity, i.e. an enzyme capable of carrying out a transpeptidation reaction in which the C-terminus of a protein is conjugated to the N-terminus of a protein by transamidation. The term includes full-length sortase proteins, e.g. full-length native sortase proteins, fragments of such sortase proteins that have sortase activity, modified (e.g. mutated) variants or derivatives of such sortase proteins or fragments thereof, as well as proteins which are not derived from native sortase proteins but which exhibit sortase activity. One of skill in the art can readily determine whether a given protein or protein fragment exhibits sortase activity, for example, by contacting the protein or protein fragment in question with an appropriate sortase substrate under conditions which allow for transpeptidation and determining whether each transpeptidation reaction product is formed.

[0027] Suitable sortases will be apparent to those skilled in the art and include, but are not limited to, sortases of the sortase A, sortase B, sortase C and sortase D type. Appropriate sortases are, for example, Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases, a nomenclature proposal for the various sortases of Gram-positive bacteria, Res. Microbiol. 156(3):289-97, 2005;Comfort D, Clubb RT, A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria., Infect. Immun., 72(5):2710-22, 2004; Chen I, Dorr BM and Liu DR, A general strategy for the evolution of bond-forming enzymes using yeast display, Proc Natl Acad. Sci. USA. 2011 Jul. 12; 108(28):11399; and Pallen, MJ; Lam, AC; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3), 97-101, the contents of each of which are incorporated herein by reference in their entireties. Any known sortase may be used as the starting enzyme in the evolutionary strategies presented herein, and the invention is not limited in this respect. For example, the invention encompasses embodiments directed to sortase A from any bacterial species or strain.Any sortase and any sortase recognition motif, for example, but not limited to, Ploegh et al., International PCT Patent Application No. PCT / US2010 / 000274, filed February 1, 2010, published August 5, 2010 as WO2010 / 087994; Ploegh et al., International Patent Application No. PCT / US2011 / 033303, filed April 20, 2011, published October 27, 2011 as WO2011 / 133303; Those skilled in the art will appreciate that the sortases and sortase recognition motifs described in U.S. Provisional Patent Application No. 61 / 662,606, filed Jun. 21, 2012; and Liu et al., U.S. Provisional Patent Application No. 61 / 880,515, filed Sep. 20, 2013, the contents of each of which are incorporated by reference in their entireties, may be used in some embodiments of the invention. The invention is not limited in this respect.

[0028] The term "sortase substrate" as used herein refers to a molecule or entity that can be used in a sortase-mediated transpeptidation reaction. Typically, a sortase uses two substrates - a substrate comprising a C-terminal sortase recognition motif and a second substrate comprising an N-terminal sortase recognition motif, and the transpeptidation reaction results in the conjugation of both substrates via a covalent bond. In the context of the present invention, the "C-terminal sortase recognition motif" is also referred to as a "sortagging donor sequence", while the term "N-terminal sortase recognition motif" is also referred to as a "sortagging acceptor sequence". In a preferred embodiment, the C-terminal and N-terminal recognition motifs are comprised in different amino acid sequences, e.g. one is the N-terminus of VSG and the other is linked to the immunogen such that there is a free carboxyl group at the end of the sortagging donor site. Several sortase recognition motifs are described herein and further suitable sortase recognition motifs will be known to those skilled in the art. For example, S. aureus sortase A preferentially catalyzes the transpeptidation reaction with a C-terminal LPXT(G / A) motif, preferably LPXTG (where X is any amino acid, and glycine cannot be a free carboxylate), and an N-terminal oligoglycine (G 1-5) (the subscript indicates the number of guanine compounds), preferably G3 or G5. Additional sortase recognition motifs will be apparent to those of skill in the art, and the invention is not limited in this respect. A sortase substrate may comprise additional moieties or entities apart from a peptidic sortase recognition motif. For example, a sortase substrate may comprise an LPXTG / A motif, the N-terminus of which is conjugated to any agent (e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label). Similarly, a sortase substrate may comprise an oligoglycine (G 1-5 ) motif, preferably G3 or G5, the C-terminus of which is conjugated to any agent, such as a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate or a detectable label. Thus, sortase substrates are not limited to proteins or peptides but include any moiety or entity conjugated to a sortase recognition motif.

[0029] The term "sortagging" in the context of referring to a donor or acceptor sequence may also be referred to as a "sortase donor sequence" or a "sortase acceptor sequence."

[0030] A "pre-ieVSG" according to the invention is an eVSG with a free N-terminal acceptor site for the addition of an immunogenic compound. For example, a pre-ieVSG is expressed on the coat of a trypanosome cell and contains a free sortase acceptor sequence on the outside of the VSG coat (e.g., accessible on the surface of the coat). Using a sortase enzyme in a sortase reaction, the pre-ieVSG can be fused to an immunogenic compound linked to a sortase donor sequence.

[0031] The term "pharmaceutically acceptable" excipient, stabilizer or carrier as used herein is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffers, lubricants, controlled release vehicles, diluents, emulsifiers, wetting agents, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent in the composition is contemplated, unless it is incompatible with the active compound. Auxiliary agents may also be incorporated into the composition.

[0032] The term "priming" or "priming immunization" or "priming immunization step" refers to a step of a method of "priming" an immunization, including the initial administration to a subject of one or more antigens in preparation for subsequent administration of the same antigen. Specifically, the term "priming" as used herein is defined as an initial immunization with an antigen that induces an immune response against a desired antigen and recalls a higher level of immune response against the desired antigen upon subsequent reimmunization with the same antigen when administered in the context of the same or a different vaccine or immunogen delivery system. Specifically, as used herein, "priming immunization" refers to the administration of a composition comprising a preparation containing an array of VSG carrier proteins linked to an antigen, preferably where the array is VSG coated. As used herein, "priming immunogenic composition or preparation" refers to such a preparation containing the VSG carrier antigen fusion of the present invention.

[0033] Another component of the methods and compositions of the invention is the use of "boosting immunization" or "boost" or "boosting immunization step", which refers to the administration of a composition that delivers an antigen or antigenic compound, coded for in the priming immunization, but using a preparation with a different form of carrier platform, preferably not an array and / or aggregate of VSG carrier protein antigen fusions as described herein, most preferably a soluble VSG carrier antigen fusion of the invention, most preferably of the same VSG, whole organism or extract, and combinations thereof. A boost may be referred to as an anamnestic response, i.e., an immune response in a previously sensitized subject. A boosting immunization or boosting immunogenic composition can include multiple doses, which may be the same or different amounts.

[0034] The term "small molecule" or "small molecule compound" or similar expressions is intended to include organic molecules with a molecular weight between 100 Daltons and 10,000 Daltons. Preferably, small molecules have a molecular weight between 200 and 5,000 Daltons. In particular, small molecules may be small chemical fragments. The term "small molecule" is not intended to include fluorescent dyes. Furthermore, the term "small molecule" is not intended to include nucleic acid polymers, large proteins and polysaccharides. However, the building blocks of nucleic acid polymers, large proteins and polysaccharides, i.e., nucleotides, amino acids and monosaccharides, as well as small oligomers thereof, may be considered as small molecules. In particular, the term "small molecule" is intended to include peptides with a size between 250 and 8,000 Daltons.

[0035] By "vaccine" is meant a composition that includes an immunogenic or antigenic compound and that, when inoculated into a subject, has the effect of stimulating a cellular immune response, including a T cell response, and / or a humoral immune response, including a B cell response that generally results in antibody production. The T cell response may be a cytotoxic T cell response against an organism expressing the antigen. However, induction of a T cell response, including other types of T cells, by the immunogens disclosed herein is also contemplated. The B cell response results in the production of antibodies that bind to the antigen. A vaccine can serve to induce an immune response in a mammal that serves to protect the mammal from disease. The terms "vaccine", "immunogen", "immunogenic polypeptide", "antigen", "antigenic compound", and the like, may be used interchangeably. The term "vaccine" does not in any way imply that the composition can completely prevent disease in a vaccinated subject or provide any particular or general level of protection against an infectious agent. In certain embodiments, a "vaccine" may be ineffective in certain subjects, but induces an immune response in other subjects.

[0036] In a first aspect, the present invention relates to a method for inducing an immune response and / or amplifying an 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, (i) the at least one priming immunization step comprises immunization of the subject with an array of immunization carrier fusions on vesicles or beads, each immunization carrier fusion consisting of a carrier protein fused to an antigenic compound, and (ii) the at least one boosting immunization step comprises immunization of the subject with the immunization carrier fusions used in (i) in non-aggregated and / or non-arrayed form, preferably in soluble form.

[0037] In the present invention, it has surprisingly been discovered that immunization procedures involving VSG proteins as carrier proteins for smaller immunogens are highly effective when arrays or high density VSG-immunogen fusions are administered during the priming step and then the same VSG-immunogen constructs are used in soluble form for one or more boosting steps.

[0038] The immunization carrier fusion is preferably an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), which comprises an antigenic compound covalently attached, optionally via a linker, to the N-terminus of the VSG carrier protein.

[0039] A preferred embodiment of the antigenic particle of the present invention includes those in which an immunogenic compound is covalently attached to the N-terminus of VSG, optionally via a linker. The immunogenic compound may be attached to VSG by any method known to those skilled in the art, such as any chemical reaction, preferably click chemistry, cross-linking or using biological entities, such as enzymes, ligases and protein-protein interactions. The term "click chemistry" in the context of the present invention refers to a chemistry tailored to rapidly and reliably create covalent bonds by linking small units that contain reactive groups (see HC Kolb, MG Finn and KB Sharpless, 2001). Any variation of such an approach may be used to attach an immunogenic compound to VSG according to the present invention.

[0040] In general, the attachment of the VSG to the immunogenic compound may include the use of any linking means or linker, which in the context of the invention disclosed herein refers to the means through which the VSG and the immunogenic compound are linked or attached to form the eVSG. The one or more linkers or linking means for linking the VSG and the immunogenic compound may be any means structurally suitable for linking the two. Exemplary linkers include the use of one or more amino acids that can be used to form peptides, in some embodiments amino acids with modified peptide backbones, small chemical scaffolds, biotin-streptavidin, organic or inorganic nanoparticles, polynucleotide sequences, peptide-nucleic acids, organic polymers, or immunoglobulin Fc domains. The means for attachment may include covalent and / or non-covalent bonds. The one or more linkers may include various sequences or other structural features that provide various functions or properties. For example, the one or more linkers may include structural elements that allow the eVSG to be derivatized.

[0041] In a preferred embodiment of the antigenic particles of the present invention, the antigenic particles comprise a linker, which is an N-terminal extension of the N-terminal sequence of wild-type VSG, preferably comprising 5 to 30 amino acids, preferably 10 to 20, more preferably about 15 amino acids. Here, the linker is used to make the N-terminus of VSG more accessible to modification, for example with an enzyme, when VSG is provided in the context of an assembled VSG coat. The linker sequence is preferably introduced into the VSG by genetic manipulation of the VSG-encoding gene. Preferably, the linker, if present, is introduced directly at the C-terminus of a signal peptide sequence that allows cell surface targeting of the VSG protein. The signal peptide is usually cleaved after transport. Thus, the term "signal peptide" as used herein refers to an amino-terminal amino acid residue that, when added to a target polypeptide, allows transport of the target polypeptide out of the cell and cleavage of the signal peptide. A preferred linker according to the present invention is the G4S linker, although any other protein linker that does not essentially interfere with VSG folding may be used in the context of the present invention. The G4S linker according to the invention disclosed herein may contain multiple consecutive 4GS, for example a preferred linker is a G4S linker which may have the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 8).

[0042] In further or alternative embodiments of the invention, the VSG may comprise any kind of sortase acceptor sequence. Such a sequence is preferably attached to the N-terminus of the VSG sequence, such that the coated surface is accessible by the sortase enzyme. If the eVSG sequence comprises an N-terminal linker, the sortase acceptor sequence is located at the N-terminus of the linker. Exemplary sortase acceptor sequences are di-alanine (AA-) or di-glycine (GG-) or any other sequence that can be used for sortase-mediated ligation.

[0043] Thus, in some embodiments of the invention, the immunogenic compound is linked to VSG, preferably by using a sortase enzyme.

[0044] Thus, in certain preferred embodiments of the invention, the antigenic compound forms a sortase-mediated covalent linkage to the carrier protein via a sortagging donor sequence, e.g. (G)3SLPSTGG, and a sortagging acceptor sequence. It may be preferred that the linkage formed by the sortagging donor sequence and the sortagging acceptor sequence comprises the sequence -(G)3SLPSTGAA- or a sortagging-functional variant thereof.

[0045] Thus, in a preferred embodiment, the immunization carrier fusion has the following (covalent) structure from N-terminus to C-terminus: antigenic compound, sortagging donor sequence, sortagging acceptor sequence, linker, VSG protein sequence.

[0046] The immunization and vaccination protocols of the present invention are particularly applicable when the antigenic compound is a small molecule drug (less than 10 kD), such as a therapeutic compound, a sugar, an allergen and / or a dependence inducer. Even more preferably, the antigenic compound is (i) delta-9-tetrahydrocannabinol (THC) or a synthetic cannabinoid, such as classical cannabinoids, non-classical cannabinoids, hybrid cannabinoids, aminoalkyl indoles and eicosanoids, such as Δ9-THC HU-210, (C8)CP47,497, 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 its derivatives, such as 3,4-methylenedioxy-methamphetamine (MDMA) ecstasy / molly, or (iii) synthetic cathinone-like alpha alpha-pyrrolidinopentiophenone (alpha-PVP), or (iv) an opioid, such as heroin, a synthetic opioid, such as fentanyl or related compounds, such as carfentanil, and other opioid analgesics, such as oxycodone (OxyContin®), hydrocodone (Vicodin®), codeine, morphine, desomorphine (krokodil), or (v) a steroid (anabolic substance), or is nicotine.

[0047] The opium alkaloids and derivatives according to the present invention are selected from opium alkaloids, phenanthrenes such as codeine, morphine, thebaine, oripavine or mixed opium alkaloids, e.g. papaveretum, esters of morphine such as diacetylmorphine (morphine dimethylacetate, heroin), nicomorphine (morphine dimethylnicotinate), dipropanoylmorphine (morphine dimethylpropionate), diacetyldihydromorphine, acetylpropionylmorphine, dmaDesomorphine, methyldesorphine, dibenzoylmorphine, ethers of morphine such as dihydrocodeine, ethylmorphine, heterocodeine.

[0048] Also included are semi-synthetic alkaloid derivatives such as buprenorphine, etorphine, hydrocodone, hydromorphone, oxycodone, oxymorphone.

[0049] Also included are synthetic opioids, such as anilidopiperidines such as fentanyl, alpha methylfentanyl, alfentanil, sufentanil, remifentanil, carfentanil, omefentanil, as well as phenylpiperidines (meperidine) such as pethidine, ketobemidone, MPPP, allylprodine, prodine, PEPAP, promedol.

[0050] Included diphenylpropylamine derivatives include propoxyphene, dextropropoxyphene, dextromoramide, bezitramide, piritramide, methadone, dipipanone, levomethadyl acetate (LAAM); difenoxin; diphenoxylate, and loperamide.

[0051] Further included are benzomorphan derivatives such as dezocine, pentazocine, phenazocine, oripavine derivatives such as buprenorphine, dihydroetorphine, etorphine, morphinan derivatives such as butorphanol, nalbuphine, levorphanol, levomethorphan, racemethorphan, others such as lephetamine, menthol (kappa-opioid agonist), meptazinol, mitragynine, tilidine, tramadol, tapentadol, eluxadoline, AP-237, 7-hydroxymitragynine.

[0052] In some embodiments, the immunogenic compound used in the context of the present invention is an allergen, or any allergy-inducing fragment thereof. "Allergen" in the context of the present invention disclosed herein means any compound capable of inducing allergy as defined below. Allergens are usually biopolymers, biological complexes of most peptides or proteins, or sugar molecules, or any fragments or monomers thereof. It is particularly preferred to use the present invention to produce antibodies against allergens in a host animal. Such antibodies can then be used as therapeutic agents against allergies, for example in human subjects suffering from allergies induced by the respective allergen.

[0053] The term "allergy" refers to any type of hypersensitivity reaction to environmental allergens that is mediated by immunological mechanisms, such as types I-IV hypersensitivity reactions, such as allergic rhinitis, asthma, and atopic dermatitis.

[0054] In another embodiment, an immunogenic compound used in the context of the present invention may be a nucleic acid or nucleobase derivative or variant against which an antibody is desired, such as an RNA or DNA nucleobase variant.

[0055] In some further or alternative embodiments of the invention, the antigenic particles include particles that are vesicles or beads. The particles are preferably selected so that they are capable of being coated with VSG protein. In some embodiments, the trypanosome is an enzyme glycophosphatidylinositol phospholipase C (GPI-PLC) negative trypanosome. Using a GPI-PLC negative blue trypanosome strain has the advantage that after inactivation of the cells, the VSG coat does not degrade but remains intact. Thus, in other further or alternative embodiments, the vesicles may be derived from non-viable, preferably non-infected, living cells, such as inactivated living cells, preferably UV cross-linked cells.

[0056] In some embodiments, an antigenic particle of the invention may comprise two or more different eVSGs on its surface, such that the antigenic particle may be used to present two or more (or three, four, five, six, etc.) different immunogenic compounds on its surface coat. For example, the different immunogenic compounds may comprise different immunogenic epitopes of one antigen, or may comprise different epitopes of different antigens.

[0057] In some other embodiments of the invention, the eVSG comprises an immunogen as one or more immunogenic amino acid sequence(s) inserted (preferably genetically) into the VSG sequence. Preferably the inserted immunogenic sequence is a heterologous sequence, most preferably inserted into the VSG sequence without causing defects in the wild-type VSG sequence. In some embodiments, the insertion is located in a surface loop of the VSG, for example in a region between two secondary structure motifs in the VSG, preferably said surface loop is located in a three-dimensional location within the VSG that is surface presented, preferably surface accessible, and capable of being presented to the immune system when the VSG is contained or assembled in the VSG coat.

[0058] In some embodiments of the invention, the immunogenic (antigenic) compound comprised in the antigenic particle is a disease-associated antigen, such as a peptide antigen, and the disease is preferably selected from a proliferative disorder, an infectious disease, an inflammatory disorder, an immunodeficiency disorder or an autoimmune disorder, or the disease is a non-communicable disease. Most preferably, the immunogenic compound is an antigen associated with an infectious disease or a proliferative disorder, and preferably the antigen is disease-associated.

[0059] A preferred antigenic particle in the context of the present invention is a live or inactivated trypanosome cell with an intact VSG coat, wherein the VSG coat comprises one or more immunogenic modified VSGs (ieVSGs), preferably a high percentage of ieVSGs (20% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, the higher the better), wherein the ieVSG comprises an eVSG protein linked at its N-terminus to an immunogenic compound. Preferably, the linkage is effected by a sortase-mediated reaction.

[0060] Further provided is a nucleic acid construct comprising an eVSG of the invention suitable for introduction into a VSG expression locus in trypanosomes. The nucleic acid construct for introduction into trypanosomes preferably has a structure as shown in any one of Figures 3A to 3C. Generally, the nucleic acid of the invention comprises the following elements in this order: a co-transposed region (CTR) of a VSG, e.g. about 1156 bp in front of the VSG2 open reading frame in the Bloodstream Expression Site-1 (BES1) of the Trypanosoma brucei (strain Lister 427) genome, immediately followed by a coding region of an eVSG according to the invention, and a telomeric seed sequence, e.g. about 150 to 250 bp, preferably 200 bp, of telomeric seed sequence. The construct may further comprise a resistance gene, e.g. blasticidin, puromycin or hygromycin, preferably following the eVSG coding region. Methods for integrating such constructs into the trypanosome genome are described in Pinger et al., 2017 (Nature Communications), the materials and methods on pages 7 and 8 of which are incorporated herein by reference.

[0061] In another preferred embodiment, the antigenic compound is a biological antigen, such as a peptide or nucleic acid immunogen, which is derived from an antigenic structure or molecule associated with a disease. A disease-associated antigen can be any antigen that is specifically and selectively expressed in cells or tissues associated with a disease, or in any pathogen that causes or is associated with a disease. Such biological antigens may be associated with cancer or tumors, or with autoimmune or inflammatory diseases. Furthermore, biological antigens may be associated with bacterial, fungal or viral diseases, or with any other unicellular pathogenic organism, or with pathogenic or parasitic animals. Typically, such biological antigens are peptides or nucleic acid molecules with specific sequences that are found only in cells or tissues associated with a pathogen or disease. Preferred peptides are small peptide fragments of proteins expressed as components of viruses, such as and preferably coronaviruses (such as COVID-19).

[0062] A preferred VSG protein for use in the context of the invention disclosed herein is the VSG from Trypanosoma brucei.

[0063] In the context of the invention disclosed herein, an array or aggregate of immunization-carrier fusions constitutes a particle coated with a plurality of immunization-particle fusions. Thus, such immunization-particle fusions are presented in an array on the surface of the particle. Preferably, an array or aggregate is present when there is more than one immunization-carrier fusion per particle, preferably more than two, more preferably more than three, or more than four, five, eight, ten, fifteen, twenty, more preferably more than fifty such molecules per particle. The density of immunization-carrier fusions on the particle will depend on the particle used. Higher density can be obtained by using larger particles, such as inactivated trypanosomes, or trypanosome coats.

[0064] Particles that can be used in the context of the present invention are vesicles or beads. Vesicles as used herein can be liposomes, or other micelles or lipid bilayer structures. Beads can be microsphere beads, such as latex, silica or paramagnetic beads, or any other nanoparticles. Beads ranging in size from 0.1 microns to 1 mm can be used in the present invention and are therefore encompassed by the term "particle" as used herein. The term particle also encompasses biological cells, as well as beads and other microscopic objects of similar size (e.g., about 0.1 to 120 microns, typically about 1 to 50 microns) or smaller (e.g., about 0.1 to 150 nm).

[0065] In a preferred embodiment of the present invention, the immunization carrier used in the method is a VSG protein, and the VSG proteins used as carriers in (i) and (ii) are from the same VSG protein type, e.g., the VSG in both (i) and (ii) is VSG3 of Trypanosoma brucei. The "same" VSG protein in this context refers to the largely identical parent VSG protein, meaning a VSG family member, e.g., the protein sequences used between the carriers used in (i) share higher sequence identity with each other as any of them for any given third VSG protein family member, compared to the carrier used in (ii). Members of the VSG protein are disclosed, for example, in Cross GAM, Kim HS, Wickstead B. Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol Biochem Parasitol. 2014;195:59-73 or at http: / / tryps.rockefeller.edu.

[0066] In some preferred embodiments, the immunization method includes at least two priming immunization steps, all of which occur before any boosting immunization steps, hi some embodiments, there may be three or four priming immunization steps before any boosting immunization steps are performed.

[0067] In certain embodiments, at least two boosting immunization steps are performed, preferably all of the boosting immunization steps being performed after (or subsequent to) any priming immunization steps.

[0068] More preferred embodiments of the present invention relate to methods in which at least two priming immunization steps and at least two boosting immunization steps are performed. Again, any priming immunization step is performed before any boosting immunization step. Some preferred embodiments may include any combination of repeated priming and boosting immunization steps.

[0069] A feature of the invention disclosed herein is that the method of the invention induces a long-lasting antibody response (preferably with an antibody titer higher than 100 μg / mL) in an immunized subject, e.g., for more than one month, preferably for more than two months. Thus, the method is preferably a method of inducing a long-lasting antibody response (preferably with an antibody titer higher than 100 μg / mL) in an immunized subject, e.g., for more than one month, preferably for more than two months.

[0070] A subject according to the invention is in certain embodiments selected from animals having an adaptive immune system, e.g., vertebrates, e.g., birds (chickens) and mammals, e.g., mice, rabbits, camels, goats, rats, dogs, cats, monkeys, hamsters or other mammals, or a human, e.g., a human patient in need of eliciting or amplifying an immune response.

[0071] In the context of the present invention, an immunogenic compound is a small molecule, a nucleic acid, or a peptide. In other embodiments, an immunogenic compound is considered to be a small molecule, a nucleic acid, a carbohydrate, a lipid, or a peptide, or any combination of these, or other chemical entities.

[0072] A preferred embodiment of the antigenic particle of the present invention includes those in which an immunogenic compound is covalently attached to the N-terminus of VSG, optionally via a linker. The immunogenic compound may be attached to VSG by any means known to those skilled in the art, such as any chemical reaction, preferably click chemistry, cross-linking or the use of biological entities, such as enzymes, ligases and protein-protein interactions. The term "click chemistry" in the context of the present invention refers to a chemistry tailored to rapidly and reliably generate covalent bonds by linking small units that contain reactive groups (see HC Kolb, MG Finn and KB Sharpless, 2001). Any variation of such an approach may be used to attach an immunogenic compound to VSG according to the present invention.

[0073] In general, the attachment of the VSG to the immunogenic compound may include the use of any linking means or linker, which in the context of the invention disclosed herein refers to the means through which the VSG and the immunogenic compound are linked or attached to form the eVSG. The one or more linkers or linking means for linking the VSG and the immunogenic compound may be any means structurally suitable for linking the two. Exemplary linkers include the use of one or more amino acids, small chemical scaffolds, biotin-streptavidin, organic or inorganic nanoparticles, polynucleotide sequences, peptide-nucleic acids, organic polymers, or immunoglobulin Fc domains, which in some embodiments may be used to form peptides with modified peptide backbones. The means for attachment may include covalent and / or non-covalent bonds. The one or more linkers may include various sequences or other structural features that provide various functions or properties. For example, the one or more linkers may include structural elements that allow the eVSG to be derivatized.

[0074] In a second aspect, the present invention relates to a method of making an antibody against an antigenic compound, comprising carrying out the method of the first aspect, optionally in any single described embodiment and variant or combination of the method of the first aspect in a non-human animal, and isolating the newly made antibodies or antibody-producing B cells from a subject (such as a non-human animal), wherein the antibody is against the antigenic compound.

[0075] In a third aspect, the present invention relates to an antibody or an antibody-producing B cell generated and / or isolated according to the method of the second aspect.

[0076] The method of the present invention in the second and third aspects provides the vaccination procedure described herein for the production of antibodies against target antigenic compounds.Surprisingly, it has been discovered that immunization by the method described herein produces a stable antibody response.Therefore, the method of the present invention is particularly useful for generating new antibodies against otherwise poorly immunogenic antigens.

[0077] The method of the second aspect preferably comprises the steps of generating hybridoma cells with the isolated B cells, and / or isolating paired heavy and light chain antibody genes and recombinantly producing them in mammalian cells.

[0078] As used herein, the term "antibody" may be understood in the broadest sense as any immunoglobulin (Ig) capable of binding to its epitope. A full-length "antibody" or "immunoglobulin" is generally a heterotetrameric glycoprotein of about 150 kDa, composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has an amino-terminal variable domain (VH) followed by three carboxy-terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interrupted by more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to cells or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Other forms of antibodies include heavy chain antibodies, which consist of only two heavy chains and lack the two light chains normally found in antibodies. Heavy chain antibodies include hcIgG (IgG-like) antibodies from camelids, such as dromedaries, camels, llamas and alpacas, and IgNAR antibodies from cartilaginous fishes, such as sharks. Yet another form of antibody is single domain antibodies (sdAbs, called nanobodies by developer Ablynx), which are antibody fragments consisting of a single monomeric variable antibody domain. Single domain antibodies are typically generated from heavy chain antibodies, but may also be derived from conventional antibodies.

[0079] Antibodies (or fragments thereof from which they can be isolated) may include, for example, chimeric, humanized, (fully) human, or hybrid antibodies with dual or multiple antigen or epitope specificity, antibody fragments and antibody subfragments, e.g., Fab, Fab' or F(ab')2 fragments, single chain antibodies (scFv), etc. (described below), e.g., hybrid fragments of any immunoglobulin or any natural, synthetic or engineered protein that acts like an antibody by binding to a specific antigen to form a complex. VSIR is an immunoglobulin-like protein and therefore it (and its variants) are - for the purposes of the present invention - not considered to be an antibody that binds to the antigenic compounds of the present invention.

[0080] Thus, in certain embodiments of the invention, the antibodies produced by the methods disclosed herein may comprise an antibody heavy chain, or an antigen-binding fragment thereof, and / or an antibody light chain, or an antigen-binding fragment thereof.

[0081] In an alternative and preferred embodiment of all ABPs of the invention, the ABP is an antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody or the antigen-binding fragment is a fragment of a monoclonal antibody.

[0082] The antibodies produced according to the present invention may be polyclonal or monoclonal.

[0083] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of antibodies that are substantially identical based on their amino acid sequence. Monoclonal antibodies are typically highly specific. Moreover, in contrast to traditional (polyclonal) antibody preparations that typically include different antibodies against different determinants (e.g., epitopes) of an antigen, each mAb is typically directed against a single determinant on the antigen. In addition to their specificity, mAbs are advantageous in that they can be synthesized by cell culture (hybridoma, recombinant cells, etc.) uncontaminated by other immunoglobulins. mAbs herein include, for example, chimeric, humanized or human antibodies, or antibody fragments.

[0084] Monoclonal antibodies according to the present invention may be prepared by methods well known to those skilled in the art. For example, mice, rats or rabbits may be immunized by the method of the first aspect. Furthermore, splenocytes of such immunized animals are collected as a pool from animals immunized several times at specific intervals, with test bleeding performed to evaluate serum antibody titers. Prepared splenocytes are either used immediately in fusion experiments or stored in liquid nitrogen for use in future fusion or cell sorting experiments followed by antibody cloning and expression of humanized monoclonal antibodies (Tiller et al., J. Immunol. Methods 2008, 329(1-2):112-124). Fusion experiments are then performed according to the procedure of Stewart and Fuller, J. Immunol. Methods 1989, 123:45-53. Supernatants from wells with growing hybrids are screened for mAb-secreting cells, for example by enzyme-linked immunosorbent assay (ELISA). ELISA positive cultures are cloned by either limiting dilution or fluorescence activated cell sorting, typically resulting in hybridomas established from single colonies. The ability of antibodies, including antibody fragments or subfragments, to bind to a specific antigen can be determined by binding assays known in the art, for example, using the antigen of interest as a binding partner.

[0085] Alternatively, the antibodies described herein may be prepared by use of XenoMouse® technology. Such mice are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of mouse immunoglobulin molecules and antibodies. In particular, preferred embodiments of the transgenic generation of mice and antibodies are disclosed in U.S. Patent Application Serial No. 08 / 759,620, filed December 3, 1996, and International Patent Applications WO 98 / 24893, published June 11, 1998, and WO 00 / 76310, published December 21, 2000. See also Mendez et al., Nature Genetics, 15:146-156 (1997). Using such technology, fully human monoclonal antibodies against a variety of antigens are produced. Essentially, XenoMouse® strains of mice are immunized with the VSG technology of the present invention, lymphocytes (such as B cells) are harvested from the hyperimmune mice, and the harvested lymphocytes are fused with a myeloid cell line to prepare immortalized hybridoma cell lines. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. Other "humanized" mice are also commercially available: for example, Medarex-HuMab mice, Kymab-Kymouse, Regeneron-Velocimmune mice, Kirin-TC mice, Trianni-Trianni mice, OmniAb-OmniMouse, Harbour Antibodies-H2L2 mice, Merus-MeMo mice. Other "humanized" species are also available: rats: OmniAb-OmniRat, OMT-UniRat, chickens: OmniAb-OmniChicken.

[0086] The term "humanized antibody" according to the present invention refers to an immunoglobulin chain or fragment thereof (such as Fab, Fab', F(ab')2, Fv, or other antigen-binding subsequence of an antibody) that contains minimal sequence (but typically still at least a portion) derived from a non-human immunoglobulin. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which CDR residues of the recipient antibody are replaced by CDR residues from a non-human species immunoglobulin (donor antibody), e.g., mouse, rat or rabbit, having the desired specificity, affinity, and capacity. Thus, at least a portion of the framework sequences of said antibody or fragment thereof may be human consensus framework sequences. In some instances, Fv framework residues of the human immunoglobulin must be replaced by corresponding non-human residues to increase specificity or affinity. Furthermore, humanized antibodies can contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These alterations (modifications) are made to further refine and maximize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, and typically at least two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the framework regions being of a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, which (e.g., human) immunoglobulin constant region has been altered (modified) (e.g., by mutation or glycoengineering) to optimize one or more properties of such region and / or to improve the function of the (e.g., therapeutic) antibody, e.g., to increase or decrease Fc effector function or increase serum half-life. Exemplary such Fc alterations (modifications) (e.g., Fc modified or Fc enhanced) are described elsewhere herein.

[0087] The term "chimeric antibody" according to the present invention refers to an antibody whose light and / or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions that are identical or homologous to the corresponding sequences of different species, e.g., mouse and human. Alternatively, the variable region genes are from a particular antibody class or subclass, while the remainder of the chains are from another antibody class or subclass of the same or different species. This also encompasses fragments of such antibodies. For example, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species may be used.

[0088] In certain such embodiments, the antibody produced according to the invention comprises an antigen-binding domain of an antibody, wherein the antigen-binding domain is that of a human antibody. Preferably, the antibody produced according to the invention comprises an antigen-binding domain of an antibody or an antigen-binding fragment thereof, which is a human antigen-binding domain, (ii) the antibody is a monoclonal antibody or the antigen-binding fragment is a fragment of a monoclonal antibody, and (iii) the antibody is a human antibody or a humanized antibody or the antigen-binding fragment is a fragment of a human antibody, a humanized antibody or a chimeric human antibody.

[0089] The light chains of human antibodies are generally classified as kappa and lambda light chains, each of which contains one variable region and one constant domain. The heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Human IgG has several subtypes, including but not limited to lgG1, lgG2, lgG3, and lgG4. Human IgM subtypes include IgM and IgM2. Human IgA subtypes include IgA1 and IgA2. In humans, IgA and IgD isotypes contain four heavy chains and four light chains, IgG and IgE isotypes contain two heavy chains and two light chains, and IgM isotypes contain 10 or 12 heavy chains and 10 or 12 light chains. The antibody according to the invention may be an IgG, IgE, IgD, IgA or IgM immunoglobulin.

[0090] In some embodiments, the antibody of the invention is an IgG antibody or a fragment thereof. In some embodiments, the antibody of the invention is an IgE antibody or a fragment thereof. In some embodiments, the antibody of the invention is an IgD antibody or a fragment thereof. In some embodiments, the antibody of the invention is an IgA antibody or a fragment thereof. In some embodiments, the antibody of the invention is an IgM antibody or a fragment thereof. Preferably, the antibody of the invention is, comprises or is derived from an IgG immunoglobulin or a fragment thereof, such as a human, human-derived IgG immunoglobulin, or a rabbit- or rat-derived IgG, and / or an IgG2 immunoglobulin or a fragment thereof. When the antibody of the invention is, comprises or is derived from a rat-derived IgG, preferably the antibody is, comprises or is derived from a rat IgG2a or IgG2b immunoglobulin. When the antibody of the invention is, comprises or is derived from a human-derived IgG, more preferably the antibody of the invention is, comprises or is derived from a human IgG1, IgG2 or IgG4, and most preferably the antibody of the invention is, comprises or is derived from a human IgG1 or IgG2.

[0091] Thus, in certain embodiments of the invention, the antibody is preferably an IgG, IgE, IgD, IgA or IgM immunoglobulin, preferably an IgG immunoglobulin.

[0092] Preferred are antibodies of the invention that comprise at least a portion of an immunoglobulin constant region (typically a human immunoglobulin constant region), where such (e.g. human) immunoglobulin constant region may be modified - for example by glycoengineering or mutation - to optimize one or more properties of such region and / or improve the function of the (e.g. therapeutic) antibody, e.g. Fc effector function may be increased or decreased, or serum half-life may be increased.

[0093] In a fourth aspect, the present invention relates to an immunization kit comprising (a) an array or aggregate of immunization carrier proteins on vesicles or beads as described in the first aspect, and (b) a non-aggregated and / or non-arrayed form, preferably a soluble form, of the immunization carrier proteins of (a), wherein the immunization carrier proteins of (a) and (b) can be fused with an antigenic compound to obtain the immunization carrier fusion as described in the first aspect.

[0094] In a fifth aspect, the present invention relates to a method for treating or preventing an adverse condition in a subject by inducing an immune response and / or amplifying an existing immune response against an antigenic compound causing or associated with the adverse condition, comprising carrying out the steps of the method of the first aspect.

[0095] In the context of the present invention, the immunization procedure of the present invention is used in the context of vaccination or other medical treatment of an individual. In that context, therefore, a further aspect of the present invention relates to a pharmaceutical composition comprising an antigenic particle according to the present invention together with a pharma- ceutically acceptable carrier and / or excipient. The pharmaceutical composition is prepared for administration to a subject for the treatment, prevention or management of a disease or disorder.

[0096] The pharmaceutical composition is preferably formulated as a vaccine composition, thus a composition suitable for vaccination in a subject in need of vaccination.

[0097] By way of example, the pharmaceutical composition of the present invention may contain between 0.1% and 100% (w / w) active ingredient, for example about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably about 1% to about 20%, about 10% to 50% or about 40% to 90%. The active ingredient of the composition of the present invention is preferably the antigenic particle of the present invention.

[0098] The term "pharmaceutically acceptable" excipient, stabilizer or carrier as used herein is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffers, lubricants, controlled release vehicles, diluents, emulsifiers, wetting agents, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent in the composition is contemplated unless it is incompatible with the active compound. Auxiliary agents may also be incorporated into the composition.

[0099] Pharmaceutical compositions of the invention (or for use in conjunction with) are typically formulated to be compatible with its intended route of administration, which include oral, parenteral, e.g., intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, intraperitoneal, transdermal (topical) and transmucosal administration.

[0100] Solutions or suspensions for parenteral, intradermal or subcutaneous application may contain a compound of (or for use with) the present invention (e.g., antigenic particles of the present invention) as well as the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial agents, such as benzyl alcohol or methylparabens, antioxidants, such as ascorbic acid or sodium hydrogen sulfate, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates or phosphates, or agents for adjusting tonicity, such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0101] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Kolliphor® EL (formerly Cre-mophor EL), and the like. (trademark), BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition for injection is typically sterile and must be fluid to the extent that easy syringability exists. It is typically stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0102] Oral compositions include a compound of (or for use with) the present invention (e.g., antigenic particles of the present invention) as well as generally an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared with a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally and swished, expectorated, or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin, an excipient such as starch or lactose, a disintegrant such as alginic acid, Primogel or corn starch, a lubricant such as magnesium stearate or Stertes, a glidant such as colloidal silicon dioxide, a sweetener such as sucrose or saccharin, or a flavouring such as peppermint, methyl salicylate or orange flavouring or compounds of a similar nature.

[0103] Additionally, the compounds of (or for use with) the present invention (e.g., antigenic particles of the present invention) may be administered rectally. Rectal compositions may be in any rectally acceptable dosage form, including, but not limited to, creams, gels, emulsions, enemas, suspensions, suppositories, and tablets. One preferred dosage form is a suppository, having a shape and size designed for introduction into the rectal opening of the human body. Suppositories usually soften, melt, or dissolve at body temperature. Suppository excipients include, but are not limited to, cacao oil (cocoa butter), glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.

[0104] For administration by inhalation, the compounds of (or for use in conjunction with) the invention (e.g., antigenic particles of the invention) are typically delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0105] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, pharmaceutical compositions can be formulated as ointments, salves, gels, or creams, as generally known in the art.

[0106] In certain embodiments, pharmaceutical compositions are formulated for sustained or controlled release of the compounds of (or for use with) the present invention (e.g., antigenic particles of the present invention). Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials may also be commercially available (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) and used as pharma- ceutically acceptable carriers. These may be prepared by methods known to those skilled in the art.

[0107] Exemplary unit dosage forms of pharmaceutical compositions containing antigenic particles of the present invention are tablets, capsules (e.g., powders, granules, microtablets or micropellets), suspensions or disposable preloaded syringes. In certain embodiments, kits for the preparation of single-dose administration units are provided. The kits may include both a first container with a dry active ingredient and a second container with an aqueous formulation. Alternatively, the kits may include single and multi-chamber preloaded syringes.

[0108] As mentioned above, the preferred pharmaceutical composition is a vaccine composition. As used herein, the term "vaccine composition" refers to an immunogenic composition that, when administered to a subject, induces protective immunity against an antigen, in this case an antigen presented in the immunogenic compound. The antibodies produced by the present invention can also be used as therapeutic agents or passive immunization.

[0109] In addition, the vaccine composition may contain one or more adjuvants. The term "adjuvant" as used herein refers to a compound that enhances or otherwise modifies or alters the resulting immune response when used in combination with a particular antigenic particle in a formulation. Modification of immune response includes enhancing and broadening the specificity of either or both antibody and cellular immune response. Modification of immune response can also mean reducing or suppressing a particular antigen-specific immune response.

[0110] Examples of known adjuvants include complete Freund's adjuvant (containing killed Mycobacterium tuberculosis, a non-specific stimulator of the immune response), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other known adjuvants include granulocyte macrophage colony stimulating factor (GM-CSF), Bacillus Calmette-Guerin (BCG), aluminum hydroxide, muramyl dipeptide (MDP) compounds such as thur-MDP and nor-MDP, muramyl tripeptide phosphatidylethanolamine (MTP-PE), RIBI adjuvant (Ribi ImmunoChem Research, Inc., Hamilton Mont.), which contains three components extracted from bacteria, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion. MF-59, Novasomes®, major histocompatibility complex (MHC) antigens are other known adjuvants.

[0111] In some embodiments, the vaccine composition comprises 2, 3, 4, 5, 6, 7, 8, or 9 or more antigenic particles, each of which comprises a different immunogenic compound derived from one or more antigens.

[0112] The substances, compositions and systems of the invention provided herein are preferably for use in the prevention, management and / or treatment of a medical condition. Accordingly, the invention relates to the use of the substances, compositions or systems disclosed herein for the manufacture of a medicament for the prevention, management and / or treatment of a medical condition. Furthermore, the invention relates to a method for the prevention, management and / or treatment of a medical condition, comprising administering an effective amount of a substance, composition or system of the invention to a subject in need of such treatment.

[0113] A medical condition that may be prevented, managed or treated by the present invention is, in a preferred embodiment, addiction to a dependence-inducing substance. In this embodiment, the immunogenic compound of the antigenic particle is a dependence-inducing substance. Without being bound by theory, the antigenic particles of the present invention provide an improved vaccination strategy, which results in a strong immune response in the subject, whereby antibodies produced in the subject in response to the antigenic particles act as a "sponge" to neutralize the dependence-inducing substance when the subject consumes such substance during relapse. The vaccination strategy of the present invention thereby reduces the positive sensation of the dependence-inducing substance and helps the subject overcome the addiction.

[0114] The medical condition that may be prevented, managed or treated by the present invention is, in another preferred embodiment, an adverse or fatal event caused by a drug contaminant. A drug contaminant is a substance that is consumed together with the preparation of an abused drug, but which often leads to an overdose, often causing severe adverse effects and even death of the subject. In this embodiment, the antigenic particles of the present invention comprise an immunogenic compound that induces an immune response against common contaminants to protect the abuser from overdosing. In such a treatment, the subject is also preferably administered an antigenic particle of the present invention specific for the abused drug to which the subject is addicted.

[0115] In a further embodiment, the present invention relates to a medical condition that is an infectious disease, and the immunogenic compound of the antigenic particle is an immunogenic compound or sequence derived from an infectious organism that causes the infectious disease. In a preferred embodiment, the disease is malaria. In other preferred aspects and embodiments, the infectious disease is a viral disease, such as a disease caused by a coronavirus, such as Covid-19. For malaria and Covid-19, known preferred antigens are well known to those skilled in the art. For example, the immunogen is a compound derived from a viral or plasmodium surface.

[0116] Another embodiment of the invention relates to cancer, wherein the immunogenic compound of the antigenic particle is a compound associated with or specific for cancer cells of the cancer.

[0117] Another embodiment of the invention relates to the creation of "delayed release depots" (SRDs) for the application of chemotherapeutic agents. An SRD is any product of an antibody or vaccination that binds to a particular chemotherapeutic compound and modifies the pharmacokinetics or other properties of the treatment for a useful purpose (e.g. reduced toxicity, extended half-life, alternative tissue targeting, etc.). Examples are anti-cancer chemotherapeutic agents (platinum, adriamycin, etc.) and the generation of an antibody or immune response from vaccination that allows the capture and "delayed release" of the chemotherapeutic agent for treatment or other useful purposes. Other examples include chemotherapeutic agents against infectious diseases or allergies, where an immunological SRD provides a useful approach. An SRD for a compound used in industrial applications would also be an embodiment of the invention.

[0118] Further embodiments of the present invention relate to aspects of neurodegenerative diseases and any product of antibodies or vaccination against specific proteins, such as Alzheimer's disease (amyloid beta (Ab) peptide, Tau), Parkinson's disease (α-synuclein), multiple tauopathies (Tau protein, microtubule associated), Huntington's disease (huntingtin with or without tandem glutamine repeats), amyotrophic lateral sclerosis (superoxide dismutase 1), spongiform encephalopathies (prion protein), familial amyloid polyneuropathy (transthyretin, wild type and mutant forms), small peptides such as those associated with migraine headaches), and any conceivable peptide or small molecule target that is deemed useful, for example but not limited to, industrially, agriculturally, in research or diagnostically.

[0119] Generally, anything in which an antibody or systemic immune response has demonstrated or could reasonably be expected to potentially demonstrate utility in a medical, diagnostic or industrial condition qualifies as an embodiment of the present invention.

[0120] The medical use of the antigenic particles of the present invention typically involves administration of the antigenic particles, e.g. in the form of a vaccine composition as described herein above, to a subject in need of the prevention, management and / or treatment of a medical condition.

[0121] Furthermore, still another aspect of the present invention is 1. A method for producing an antibody capable of binding to an immunogenic compound, comprising the steps of: Providing an antigenic particle according to the invention, wherein the immunogenic compound of the antigenic particle is an immunogenic compound or an immunogenic part thereof to which the antibody to be generated can bind; immunizing an antibody producing human or non-human animal with the antigenic particles; Isolating immune cells from the immunized human or animal that produce antibodies against the immunogen; and Optionally, isolating the antibody so produced from said cells. The present invention relates to a method comprising the steps of:

[0122] In the context of the invention disclosed herein, the non-human animal is preferably selected from a mouse, rabbit, camel, chicken, goat, rat, dog, cat, monkey, hamster or other mammal.

[0123] A further aspect relates to a method of vaccinating a subject in need of an enhanced immune response specific for an immunogenic compound, said method comprising administering to the subject an amount of antigenic particles according to the invention sufficient to induce in the subject an immune response against the immunogenic compound, wherein the immunogenic compound comprised in the antigenic particles is identical to or an immunogenic part of the immunogenic compound to which the enhanced immune response is specific.Preferred is a method of preventing, managing or treating a medical condition, wherein the immunogenic compound is associated with the medical condition.

[0124] Such an adverse condition is, for example, intoxication, and the antigenic compound is an intoxicant.

[0125] It should be understood that the application of the teachings of the present invention to a particular problem or environment and the inclusion of modifications or additional features of the present invention (e.g., additional aspects and embodiments) will be within the capabilities of one of ordinary skill in the art in light of the teachings contained herein.

[0126] Unless the context indicates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0127] All references, patents and publications cited herein are hereby incorporated by reference in their entirety.

[0128] The sequence shows:

[0129] SEQ ID NO:1>Tb427VSG-1: GB accession X56761.2|Trypanosoma brucei|Lister 427|Mutant surface glycoprotein MITat1.1 (Lister 427-1|Not entirely constructed by us)|Source=GenBank download 170507|Protein length=492

[0130] TIFF0007656627000001.tif36169TIFF0007656627000002.tif28168

[0131] SEQ ID NO:2>Tb427VSG-2: GB accession X56762.1|Trypanosoma brucei|Lister 427|Mutated surface glycoprotein MITat1.2 (Lister 427-2|Identical to our construction)|Source=GenBank download 170507|Protein length=476

[0132] TIFF0007656627000003.tif59169

[0133] SEQ ID NO:3>Tb427VSG-3: GB accession AY935575.1|Trypanosoma brucei|Lister 427|Mutated surface glycoprotein MITat1.3 (Lister 427-3|Identical to our construction)|Source=GenBank download 170507|Protein length=509

[0134] TIFF0007656627000004.tif59169

[0135] SEQ ID NO:4>Tb427VSG-13: GB accession AY935576.1|Trypanosoma brucei|Lister 427|Mutant surface glycoprotein MITat1.13 (Lister 427-13|Not entirely constructed by us)|Source=GenBank download 170507|Protein length=499

[0136] TIFF0007656627000005.tif21168TIFF0007656627000006.tif40169

[0137] SEQ ID NO:5>X56767.1|Trypanosoma brucei|ILTat1|mRNA variant surface protein ILTat1.24|Source=GenBank download 170421|Protein length=514

[0138] TIFF0007656627000007.tif60169 EXAMPLES

[0139] Certain aspects and embodiments of the invention are described herein, by way of example, with reference to the description, figures and tables herein. Such examples of methods, uses and other aspects of the invention are merely representative and should not be construed as limiting the scope of the invention solely to such representative examples.

[0140] The examples show:

[0141] [Example 1] Drug-modified VSG coating

[0142] The inventors have generated tools to derivatize the dense and homogenous surface coat of African trypanosomes (Trypanosoma brucei) for use as a presentation platform for (any) antigen against which antibodies need to be generated. These tools consist of: (a) Specific vectors for efficient replacement of an expressed VSG (VSG2) with any other VSG of interest (see below); FIG. 1 contains maps of two such plasmid vectors. (b) A particular set of VSGs of interest: these contain extended N-termini that are accessible to the enzyme sortase (here from Streptococcus pyogenes). N-terminal accessibility is determined by (i) the relative position on the VSG (structurally determined - FIG. 2 includes VSG2 and VSG3, whose N-termini are accessible, and also VSG13, whose N-terminus is inaccessible due to steric hindrance, for comparison). This is also determined by (ii) the starting amino acid, which must be Ala-Ala for the particular sortase used (or Gly-Gly for sortases from other organisms). (c) VSGs fulfilling these criteria are modified into the modified Lister 427 strain of Trypanosoma brucei by replacing active VSGs (see (a) above - Figure 4 contains the amino acid sequences of three such VSGs: VSG3 (A), VSG2 (B) and ILTat1.24 (C). It is worth mentioning that VSG3, which is modified for sortagging purposes, contains a mutation (S317A) that excludes a native glycosylation event that we have recently shown to be immunosuppressive - Pinger et al., Nat. Microbiology, 2018). (d) The modification of the Lister 427 strain of Trypanosoma brucei consists of a genetic deletion of endogenous glycophosphatidylinositol phospholipase C (GPI-PLC), the enzyme that "sheds" VSG from the surface of dying cells (this is important for generating Trypanosoma brucei that can be used as a vaccine display platform because unless GPI-PLC is removed from the genome (i) switching and loss of the modified VSG is not possible, and (ii) any form of parasite inactivation (e.g. UV irradiation), which is important for eliminating infectivity, also causes the breakdown of the VSG coat and the cell itself (once the VSG is shed by the action of GPI-PLC, the VSG coat disintegrates and the cell lyses - Figure 5 illustrates the concept)).

[0143] The method disclosed herein relies on Trypanosoma brucei eliciting a neutralizing (and long-lasting) antibody response against its VSG coat, "hijacking" its natural ability to produce antibodies at will. We do this by modifying the VSG coat with any peptide epitope / antigen, as well as sugars, lipids or small molecules, and then using the modified VSG coat as a vaccine carrier. Specifically, we use the enzyme sortase A to covalently ligate any moiety to a VSG coat engineered to have an N-terminal sortase acceptor sequence (Figures 4 and 6).

[0144] Therefore, we produce His-tagged sortase A from S. pyogenes in E. coli using a plasmid (pSpSortA-pET28a) containing the S. pyogenes sortase A expression construct. The plasmid is transformed into BL21 DE3 cells (Life Technologies C6000-03). A colony from this transformation is used to inoculate a large culture of LB medium (Sigma-Aldrich, L3022-1KG), which is then grown at 37°C with shaking to an optical density (OD600) of 0.4 to 0.8. The culture is induced with 1 mM IPTG, grown for an additional 3 to 4 hours, and harvested by centrifugation. The cell pellet is resuspended in TBS / imidazole (20 mM Tris, 150 mM NaCl, 20 mM imidazole) and lysed using an EmulsiFlex-C5 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 particles. 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 to 35 ml of elution buffer (20 mM Tris, 300 mM NaCl, 200 mM imidazole, 5 mM 2-ME). The protein-containing samples are then pooled and dialyzed in dialysis buffer (20 mM Tris, 150 mM NaCl, 1 mM DTT). The resulting samples are concentrated using centrifugal filter units (Amicon Ultra-15, 10,000 NMWL, Merck Millipore), aliquoted and stored at -80°C for future use.

[0145] The sortagging reaction is performed as follows: a mixture of sortagging solution containing 100 μM purified sortase A and 300-600 μM sortagable peptide in HMI-9 medium is incubated on ice for 30 to 60 minutes. (Sortagable peptides include any peptide with a C-terminal sortase donor sequence, LPSTGG, that can be conjugated at the N-terminus to another moiety; the moiety can be a fluorophore such as 6-FAM, a small molecule such as 4-hydroxy-3-nitrophenylacetyl (NP) hapten, or other small molecule that is a drug of abuse (e.g., fentanyl, etc.). GPI-PLC-negative Trypanosoma brucei cells expressing modified VSG are then pelleted, resuspended in sortagging mixture, and incubated on an end-over-end rotator for 60 minutes at 4 °C. The cells are then pelleted, washed once with HMI-9 medium, pelleted again, and finally resuspended in HMI-9 medium (Hirumi and Hirumi, J. Parasitology, 1989). The efficiency of sortagging can be determined by direct FACS analysis (e.g., for fluorophores such as 6-FAM) or fluorescence microscopy, or by using specific monoclonal antibodies that bind to moieties that modify VSG (Figure 7 includes examples for 6-FAM, NP hapten, and fentanyl hapten).

[0146] In proof-of-principle studies, this approach was used to generate antibodies against a small molecule hapten (4-hydroxy-3-nitrophenylacetyl or NP) that were (a) robust (compared to NP-CGG in Alum adjuvant) and (b) of consistent quality (Figure 8).

[0147] This approach can be used for a wide range of other small molecules (e.g., drugs of abuse such as cocaine, nicotine, fentanyl, carfentanil, tramadol, ketamine, etc., but also chemotherapeutic drugs such as platinum, adriamycin, etc., and also small molecules that are industrial by-products of chemical reactions), toxins that mediate allergic reactions (e.g., aflatoxins, etc.), specific peptides that serve as important epitopes for infectious diseases (e.g., Plasmodium-derived peptides), glycosylated or lipidated peptides (e.g., aberrantly glycosylated mucin peptides that are considered targets for anti-cancer vaccines, etc.).

[0148] From the perspective of an anti-fentanyl (anti-overdose) vaccine, the primary focus is to use this system to vaccinate "at risk" individuals (defined as individuals who are regular users or substance abusers but not yet addicted / chemically dependent, or addicted individuals leaving a rehabilitation center as active protection against overdose, which in addictive cases typically occurs within the first two months of leaving rehab). Proof of principle that this has been achieved using the approach herein is shown in Figures 9 and 10. Furthermore, when this approach is combined with repertoire analysis (Figure 11), a variety of anti-fentanyl monoclonal antibodies of diverse affinities (directly accessible and ready to be reconstituted from paired immunoglobulin heavy and light chain sequences generated as a result of repertoire sequencing - Figure 11) are produced. Such monoclonal antibody "sponges" could be used directly in therapeutic applications (e.g., infusing anti-fentanyl antibodies in conjunction with methadone maintenance therapy to suppress bioavailability as well as cravings and accelerate treatment outcomes, or injecting into the ER to blunt the effects of an overdose along with naloxone (which acts rapidly by antagonizing fentanyl's binding to opioid receptors, but is metabolized more quickly than fentanyl, allowing for a delay in addiction).

[0149] Methodology: The ability of trypanosomes to stimulate robust immune responses in infected individuals (responses that are long-lasting and neutralizing) is well documented. The present invention makes this possible, at least in part, by the discovery that the VSG proteins of trypanosomes are permissive for the highly efficient presentation of exogenous moieties on their surface using a bacterial transpeptidase sortase-based system (hereafter "sortagging").

[0150] Specifically, a sortase-specific sortase acceptor sequence (Ala-Ala for sortase A from Streptococcus pyogenes and Gly-Gly for sortase A from Staphylococcus aureus) may be added to the exposed N-terminal portion of the VSG protein, which remains accessible to the sortase when transported to the surface of the trypanosome (see Figures 2 and 6). Note that although VSGs start with a methionine in the signal peptide, the peptide is cleaved during maturation, and therefore the mature VSG sequence does not start with a methionine. For example, both VSG2 and VSG3 (preferred VSG variants) start with Ala-Ala, but the exact start amino acid must be determined empirically (using Edman degradation, which we performed on both VSGs). Finally, an endogenous Ala-Ala is present, but this is not accessible to the sortase (and requires a short N-terminal extension, as shown in Figure 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 from Streptococcus pyogenes it is LPXTGG), which may be added to a small molecule or other moiety bearing a reactive group (here we use 6-FAM or the hapten 4-hydroxy-3-nitrophenylacetyl, abbreviated as NP or Fen-, FIG. 7).

[0151] For fentanyl, the N-terminus of the sortase sequence is linked to the fentanyl hapten via an amide bond at the position located in Figure 7D. Most moieties may be derivatized, and derivatization that maintains the antigenicity of the small molecule-peptide conjugate (e.g., NP-GGGSLPSTGG) must be empirically determined (for example, this has been done through trial and error for nicotine and other small molecules, e.g., NicVax). The sortase then ligates the NP-bearing peptide to the exposed N-terminus of VSG on the surface of the trypanosome (we demonstrate this using an anti-small molecule antibody, Figure 7). It is also possible to insert a sortase signal sequence into the loop of the VSG (as has been done for the FLAG peptide in Stavropoulos and Papavasiliou, 2010). However, in such situations, it is prudent to increase specificity using a sortase donor sequence (e.g., LPXTGG) in the VSG loop and a sortase acceptor sequence (e.g., Ala-Ala) on the modified small molecule. Due to the high density of the coat of VSG on the surface of trypanosomes, small molecules are then presented on the surface at high density. When trypanosomes are injected into a mammalian host, the small molecule conjugated VSG coat is exposed to the host immune system, which initiates a strong and specific priming immune response against the exposed hapten (e.g., NP) similar to that against VSG in natural infection (Figure 8). Boosting can then be achieved either by the hapten-VSG conjugate on the entire coat (Figure 8) or by its formulations (e.g., soluble haptenized VSG as well as other formulations), achieving the scalability unique to this vaccine platform.

[0152] Interestingly, the primary responses elicited by different VSGs are not cross-reactive (i.e., antibodies against VSG2 do not cross-react with VSG3, etc., Pinger et al., Nat. Communications, 2017). This suggests that each particular VSG elicits a unique subset of B cell specificities (i.e., a unique repertoire) that may be stronger or weaker against a particular set of small molecule haptens. In the context of the present invention, for the elicitation of optimal anti-hapten responses, Selecting a specific VSG for a specific hapten (Some VSGs are better platforms for certain haptens - Figure 7). For example, good anti-HA (pan-influenza) antibodies require engagement of IGHV1-69, a "rare" IGH within the overall repertoire. Thus, if one wishes to use this method to elicit polyspecific anti-influenza antisera (e.g. in a "pan-flu" vaccine), it may be desirable to engage VSGs that elicit IGHV1-69. Direct evidence that VSGs elicit distinct B cell repertoires (and that this system may provide a range of different platforms depending on the preferences of the elicited B cells) is shown in Figure 11.

[0153] Biosafety concerns (e.g. disease causation) as well as the need to inhibit the natural switch from haptenized VSG dictate that the derivatized trypanosome coat be inactivated (and therefore unable to cause infection). We achieved this via UV crosslinking of trypanosomes that lack the enzyme glycophosphatidylinositol phospholipase C (GPI-PLC) and are therefore dead, but have an intact VSG coat (trypanosomes wild-type for GPI-PLC collapse after UV inactivation because GPI-PLC cleaves the GPI linkage of each VSG from the membrane and sheds the coat) (illustrated in Figure 5). UV crosslinking (of GPI-PLC-negative trypanosomes) was performed by pelleting cells from culture, washing with irradiation buffer (PBS supplemented with 55 mM glucose) and then incubating for 10 min in the same buffer. 7Irradiation is achieved by resuspending the cells to a density of 15 × 10 cells / ml. 1 ml of this suspension is then dispensed into each well of a 6-well tissue culture plate (Thermoscientific, 150239). The plate is UV irradiated for 8 cycles, each cycle for 30 seconds (30S) using a UVP crosslinker (Analytik Jena). The plate is rotated between irradiation cycles to ensure that the trypanosomes are evenly irradiated. The irradiated cells are then diluted to 15 × 10 6 Resuspend the cells at a concentration of 3 x 10 cells / ml. 6 Trypanosoma brucei) may be injected intraperitoneally or subcutaneously into mice.

[0154] Overall, using this inactivation protocol and sortagable VSG, we have created an optimal and flexible platform for immunogenic presentation of antigenic determinants towards antibody generation against small molecule haptens and peptides. This can be extended to a wide range of antigenic entities (e.g. lipids, nucleic acids, etc.). Proof of principle for antibody generation against small molecules (e.g. NP) is shown in Figure 9. Proof of principle that such antibodies can be produced against fentanyl and protection against intoxication when generated is shown in Figures 9 and 10. A schematic version of the overall method for the small molecule 6-FAM is illustrated in Figure 6.

[0155] An example of a method for incorporating a modified VSG of the invention into the Trypanosoma brucei genome is shown in FIG.

[0156] Generalizability of the approach: For the purposes of this application, we focus on active immunotherapy against fentanyl, a contaminant in synthetic heroin and the cause of the majority of drug overdoses in the United States. This is because we have the tools already available (fentanyl haptenized to LPXTGG so that it can be sortagged, anti-fentanyl antibodies to verify sortagability - from M. Pravetoni). However, it should be clear that this approach can be readily applied to the generation of effective antibodies against other drugs and drug metabolites (e.g. acetaminophen metabolites that cause liver toxicity, small molecules that are toxins responsible for anaphylactic shock in certain food allergies, etc.). This approach can also be used for haptenization with pathogen-derived peptides (e.g., the NANP tandem repeats, a major antigenic determinant of the circumsporozoite protein of Plasmodium falciparum) or aberrantly glycosylated peptides specific to cancer cells (e.g., mucins) that can be used as anti-cancer vaccines (PMID: 20403708).

[0157] [Example 2] A unique prime-boost combination for generating high-quality antibody responses and B cell memory The immunization scheme developed in the experiments of Figures 8 and 9 above was further tested. The present invention is based on a different method for prime and boost, which does not require an adjuvant but appears to induce cognate T cell help to small molecules in a physiological manner. Specifically, the inventors used a hapten-carrier combination (e.g., fentanyl covalently bound to trypanosome VSG proteins) in a prime-boost combination with two completely separate biophysical entities: the whole trypanosome VSG coat (very dense and repetitive micrometer-sized particles), and then this time the same carrier but in a soluble form (Figure 12).

[0158] It was found that the haptenized VSG particles, regardless of their size, present a highly immunodominant surface capable of eliciting a highly specific antibody repertoire and a predominantly IgM response (Figure 13A). Conversely, it was found that the same moieties used as soluble forms during boosting are capable of eliciting high IgG titers (proportionally equivalent to the carrier, both in terms of hapten and memory) (Figure 13B). Neither the priming nor the boosting step requires an adjuvant, suggesting that the present invention is based on a previously unknown physiological mechanism that generates high antibody titers and memory. Moreover, the amount used is about 250-fold less than that used in standard protocols (e.g., 100 μg NP-CGG per mouse), resulting in long-lasting (e.g., increasing even one month after the first boost) titers (Figure 13C).

[0159] [Example 3] Specific Priming-Boosting Protocols - Dosing and Timing Here, typically, two doses were administered within 30 days (days 0 and 30) as a priming step. This double step, although not essential, may help activate rare B cell receptors from the germline repertoire. We then boosted on day 60, or on days 60 and 90. The boost on day 60 results in a recall IgG response that continues to increase in titer for the next 30 days (Figure 13C).

[0160] As mentioned, doses about 250 times lower than those delivered by standard methods are sufficient in the protocol of the present invention. However, dose escalation (increase) in the priming step is not necessarily beneficial and may even be potentially harmful (injection of material presenting more than 50 million cells results in reduced titers compared to injections presenting 3 or 10 million cells - FIG. 14). The optimized protocol included a priming dose presenting about 5 million cells per about 20 g body weight, which resulted in a substantial IgG response (about 15% of total IgG in mice (2.38 mg / ml total IgG in mice (Klein-Schneegans et al., 1989)) - FIG. 15). Therefore, the two-step prime / boost process must be used in higher organisms as well, starting with small doses as mentioned above.

[0161] In the protocol below, details regarding successful immunization schedules are provided, using fentanyl as an example.

[0162] Six 6- to 8-week-old female C57BL / 6J mice per group were immunized subcutaneously (sc) with 5E6 UV-irradiated, sortagged VSG3-fentanyl cells (VSG3-Fen) in 200 μl PBS on days 0 and 30. As controls, six mice were left unimmunized or injected with untagged, UV-irradiated VSG3 carrier.

[0163] Two boosts were given using unadjuvanted soluble VSG3 or VSG3-Fen in PBS on days 60 and 90. Blood samples were taken from the facial vein 4 days before immunization began (day -4), 2 days before the first boost (day 58) and 8 days after the first boost (day 68), and 2 days before and 8 days after the second boost (days 88 and 98) with soluble VSG3 or VSG3-Fen protein (this material can be prepared in advance, concentrated and frozen in PBS; it can be thawed and diluted in PBS at the correct dose prior to injection).

[0164] The results are shown in Figure 16.

[0165] Specific IgG antibody titers in serum against both the Fen-hapten and the VSG3 carrier protein were measured by ELISA using 96-well plates coated with BSA-Fen conjugate or purified VSG3.

[0166] [Table 1]

[0167] [Example 4] Comparison of vesicles and UV-treated coats To compare the long-term efficacy of vaccination using vesicles with the UV-treated coat, immunizations were performed as described in Example 3 using vesicles containing VSG3-Fen against the UV-treated trypanosome coat.

[0168] Anti-fentanyl IgG titers (normalized AUC values) were determined over a time course including measurements on days 2, 12, 22, 40 and 50; see FIG. 17. Priming on the left side was performed with UV-treated trypanosome coat and vesicles were used on the right side. Boosting was performed in both groups using soluble VSG as previously described. A significantly stronger increase in IgG titers was found for vesicles. Therefore, vaccination with vesicles is clearly superior to vaccination with UV-treated coats, and vesicles are more suitable for GMP manufacturing and have a longer shelf life. The present invention provides the following: 1. A method for inducing an immune response and / or amplifying an existing immune response to 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; (i) at least one priming immunization step comprises immunizing a mammal with an array of immunization-carrier fusions on vesicles or beads, each immunization-carrier fusion being composed of a carrier protein fused to an antigenic compound; (ii) at least one boosting immunization step comprises immunizing a mammal with the immunization carrier fusion used in (i) in a non-aggregated, preferably soluble, form; method. 2. The method of claim 1, wherein the immunization carrier fusion is an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), the eVSG comprising an antigenic compound covalently linked to the N-terminus of the VSG carrier protein, optionally via a linker. 3. A method according to claim 1 or 2, wherein the immunization carrier fusion has the following (covalent) structures from N-terminus to C-terminus: antigenic compound, sortagging donor sequence, sortagging acceptor sequence, linker, VSG protein sequence. 4. The antigenic compound is a disease-associated antigen, such as a cancer antigen, an allergen, or a viral immunogen; or (i) delta-9-tetrahydrocannabinol (THC) or a synthetic cannabinoid, such as a classical cannabinoid, a non-classical cannabinoid, a hybrid cannabinoid, an aminoalkyl indoles and eicosanoids, such as Δ9-THC HU-210, (C8) CP47,497, 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 its derivatives, such as 3,4-methylenedioxy-methamphetamine (MDMA) ecstasy / molly; or (iii) a synthetic cathinone-like alpha-pyrrolidinopenes. thiophenone (alpha-PVP), or (iv) an opioid, such as heroin, a synthetic opioid, such as fentanyl or related compounds, such as carfentanil, and other opioid analgesics, such as oxycodone (OxyContin®), hydrocodone (Vicodin®), codeine, morphine, desomorphine (krokodil), or (v) a steroid (anabolic substance), or nicotine. 5. The method according to any one of 1 to 4 above, wherein the carrier protein is the VSG protein derived from Trypanosoma brucei. 6. A method according to any one of 1 to 5 above, wherein the vesicles or beads are coated with more than one immunized particle fusion, preferably more than 10 immunized particle fusions per particle. 7. A method according to any one of 1 to 6 above, wherein the immunisation carrier used in the method is a VSG protein, and the VSG proteins used as carriers in (i) and (ii) are from the same VSG protein type, e.g. the VSG in both (i) and (ii) is VSG3 of Trypanosoma brucei. 8. The method according to any one of 1 to 7 above, comprising at least two priming immunization steps, all of which are performed before any of the boosting immunization steps. 9. The method according to any one of 1 to 8 above, comprising at least two boosting immunization steps, all of which are performed after any of the priming immunization steps. 10. A method according to any one of 1 to 9 above, wherein the subject is an animal having an adaptive immune system, such as a vertebrate, such as a bird (chicken) and a mammal, such as a mouse, rabbit, camel, goat, rat, dog, cat, monkey, hamster or other mammal, or a human, such as a human patient in need of induction or amplification of an immune response. 11. A non-therapeutic method for producing an antibody against an antigenic compound, comprising the steps of performing a method according to any one of 1 to 10 above in a non-human animal, and isolating the antibody against the antigenic compound or an antibody-producing B cell from the non-human animal. 12. An antibody or an antibody-producing B cell generated and / or isolated according to the method described in 11 above. 13. An immunization kit comprising (a) an aggregate of an immunization carrier protein on a vesicle or bead as described in any one of 1 to 10 above, and (b) a non-aggregated, preferably soluble, form of the immunization carrier protein of (a), wherein the immunization carrier proteins of (a) and (b) can be fused with an antigenic compound to obtain an immunization carrier fusion as described in any one of 1 to 10 above. 14. An immunizing-carrier fusion for use in a method for treating or preventing a deleterious condition in a subject by inducing an immune response and / or amplifying an existing immune response to an antigenic compound causing or associated with the deleterious condition, wherein treatment comprises performing in the subject the steps of the method according to any one of 1 to 10 above.

Claims

1. 1. An immunization-carrier fusion for use in a method of treating or preventing an adverse condition in a subject by eliciting an immune response and / or amplifying a pre-existing immune response to an antigenic compound causing or associated with the adverse condition, comprising: (i) each immunization carrier fusion is composed of a carrier protein fused to an antigenic compound and is used for at least one priming immunization step comprising immunizing a subject with an array of immunization carrier fusions on a vesicle; and (ii) for at least one boosting immunization step comprising immunizing a subject with a non-aggregated form of the immunization carrier fusion used in (i); The immunization carrier fusion is an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), the eVSG comprising an antigenic compound covalently attached, optionally via a linker, to the N-terminus of the VSG carrier protein; The antigenic particles are vesicles derived from UV cross-linked inactivated trypanosomes, and the trypanosomes are trypanosomes negative for the enzyme glycophosphatidylinositol phospholipase C, Immunization carrier fusions.

2. The immunizing carrier fusion of claim 1, wherein the non-aggregated form in step (ii) is a soluble non-aggregated form.

3. The immunization-carrier fusion of claim 1 or 2, having the following (covalent) structures from N-terminus to C-terminus: antigenic compound, sortagging donor sequence, sortagging acceptor sequence, linker, VSG protein sequence.

4. the antigenic compound is a disease-associated antigen or (i) delta-9-tetrahydrocannabinol (THC) or a synthetic cannabinoid, or (ii) methamphetamine and its derivatives which are 3,4-methylenedioxy-methamphetamine (MDMA) ecstasy / molly; or (iii) a synthetic cathinone-like alpha-pyrrolidinopentiophenone (alpha-PVP); or (iv) an opioid, synthetic opioid, and other opioid analgesics; or (v) a dependence-inducing substance selected from steroids (anabolic substances); or nicotine.

5. 5. The immunizing carrier fusion of claim 4, wherein the disease-associated antigen is a cancer antigen, an allergen, or a viral immunogen.

6. 5. The immunization carrier fusion of claim 4, wherein the synthetic cannabinoid is a classical cannabinoid, a non-classical cannabinoid, a hybrid cannabinoid, an aminoalkyl indole or an eicosanoid.

7. 5. The immunization-carrier fusion of claim 4, wherein the synthetic cannabinoid is selected from A9-THC HU-210, (C8)CP47,497, JWH-018, AM-2201 (fluorinated JWH-018), UR-144, XLR-11 (fluorinated UR-144), APICA, STS-135 (fluorinated APICA), AB-PINACA, PB-22, and 5F-PB-22 (fluorinated PB-22).

8. 5. The immunized-carrier fusion of claim 4, wherein the opioids, synthetic opioids, and other opioid analgesics are selected from heroin, fentanyl, carfentanil, oxycodone (OxyContin®), hydrocodone (Vicodin®), codeine, morphine, and desomorphine (krokodil).

9. 9. The immunizing carrier fusion of any one of claims 1 to 8, wherein the carrier protein is the VSG protein from Trypanosoma brucei.

10. 10. The immunizing carrier fusion of any one of claims 1 to 9, wherein the vesicle is coated with more than one immunizing particle fusion per particle.

11. The immunizing-carrier fusion of claim 10, wherein the vesicles are coated with more than 10 immunizing-particle fusions per particle.

12. The immunization carrier fusion of any one of claims 1 to 11, wherein the immunization carrier used in the method is a VSG protein, and the VSG proteins used as carriers in (i) and (ii) are from the same VSG protein type.

13. 13. The immunization carrier fusion of claim 12, wherein the VSG in both (i) and (ii) is VSG3 of Trypanosoma brucei.

14. 14. The immunization carrier fusion of any one of claims 1 to 13, wherein at least two priming immunization steps are performed, all of the priming immunization steps being performed before any of the boosting immunization steps.

15. 15. The immunization carrier fusion of any one of claims 1 to 14, wherein at least two boosting immunization steps are performed, all of the boosting immunization steps being performed after any of the priming immunization steps.

16. 16. The immunizing carrier fusion of any one of claims 1 to 15, wherein the subject is selected from an animal or a human having an adaptive immune system.

17. 17. The immunizing carrier fusion of claim 16, wherein the animal having an adaptive immune system is a vertebrate, an avian, or a mammal.

18. 17. The immunizing carrier fusion of claim 16, wherein the mammal is selected from the group consisting of a mouse, rabbit, camel, goat, rat, dog, cat, monkey, hamster or other mammal, or the human is a human patient in need of eliciting or amplifying an immune response.

19. A non-therapeutic method for generating antibodies against an antigenic compound, 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, (i) at least one priming immunization step comprises immunizing a non-human animal with an array of immunization-carrier fusions on a vesicle, each immunization-carrier fusion being composed of a carrier protein fused to an antigenic compound; (ii) at least one boosting immunization step comprises immunizing a non-human animal with the immunization carrier fusion used in (i) in a non-aggregated form; isolating antibodies or antibody-producing B cells against an antigenic compound from the non-human animal; The immunization carrier fusion is an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), the eVSG comprising an antigenic compound covalently attached, optionally via a linker, to the N-terminus of the VSG carrier protein; The antigenic particles are vesicles derived from UV cross-linked inactivated trypanosomes, and the trypanosomes are trypanosomes negative for the enzyme glycophosphatidylinositol phospholipase C, method.

20. An immunization kit comprising: (a) an aggregate of an immunization carrier protein on a vesicle according to any one of claims 1 to 18; and (b) a non-aggregated form of the immunization carrier protein of (a), wherein the immunization carrier proteins of (a) and (b) can be fused with an antigenic compound to obtain an immunization carrier fusion according to any one of claims 1 to 18, The immunization carrier fusion is an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), the eVSG comprising an antigenic compound covalently attached, optionally via a linker, to the N-terminus of the VSG carrier protein; The antigenic particles are vesicles derived from UV cross-linked inactivated trypanosomes, and the trypanosomes are trypanosomes negative for the enzyme glycophosphatidylinositol phospholipase C, Immunization kit.

21. 21. The immunization kit of claim 20, wherein the non-aggregated form is a soluble form.

22. 20. A pharmaceutical composition comprising the immunizing carrier fusion of any one of claims 1 to 18.

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