Topical therapy for regression and melanoma prevention of congenital giant nevi.

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.

CDR L3 Loop Rearrangement Switches Multispecific SPE-7 IgE Antibody From Hapten to Protein Binding.

The monoclonal IgE antibody SPE-7 was originally raised against a 2,4-dinitrophenyl (DNP) target. Through its ability to adopt multiple conformations, the antibody is capable of binding to a diverse range of small haptens and large proteins. The present study examines a dataset of experimentally determined crystal structures of the SPE-7 antibody to gain insight into the mechanisms that contribute to its multispecificity. With the emergence of more and more therapeutic antibodies against a huge repertoire of different targets, our research could be of great interest for future drug development. We are able to discriminate between the different paratope-binding states in the conformational ensembles obtained by enhanced sampling molecular dynamics simulations, and to calculate their transition timescales and state probabilities. Furthermore, we describe the key residues responsible for discriminating between the different binding capacities and identify a tryptophan in a central position of the CDR L3 loop as the residue of greatest interest. The overall dynamics of the paratope appear to be mainly influenced by the CDR L3 and CDR L1 loops.

Microchimeric cells promote production of rheumatoid arthritis-specific autoantibodies.

BACKGROUND: Women are more likely to develop autoimmune diseases than men. Contribution from microchimerism (Mc) has been proposed, as women naturally acquire Mc from more sources than men because of pregnancy. Women with Rheumatoid Arthritis (RA) who lack RA-associated HLA alleles have been found to harbor Mc with RA-associated HLA alleles in higher amounts than healthy women in prior work. However, an immunological impact of Mc remains to be elucidated. OBJECTIVES: To test the hypothesis that Mc with RA-risk associated HLA alleles can result in the production of RA-associated autoantibodies, when host genetic risk is absent. METHODS: DBA/2 mice are unable to produce RA-specific anti-citrullinated autoantibodies (ACPAs) after immunization with the enzyme peptidyl arginine deiminase (PAD) in a previously developed model. DBA/2 females were mated with C57BL/6 males humanized to express HLA-DR4, which is associated with RA-risk and production of ACPAs, to evaluate DR4+ fetal Mc contribution. Next, DBA/2 females born of heterozygous DR4+/- mothers were evaluated for DR4+ Mc of maternal or littermate origin. Finally, DBA/2 females from DR4+/- mothers were crossed with DR4+ males, to evaluate the contribution of any Mc source to ACPA production. RESULTS: After PAD immunization, between 20 % and 43 % of DBA/2 females (otherwise unable to produce ACPAs) had detectable ACPAs (CCP2 kit) after exposure to sources of Mc with RA-associated HLA alleles, compared to 0 % of unmated/unexposed DBA/2 females. Further the microchimeric origin of the autoantibodies was confirmed by detecting a C57BL/6-specific immunoglobulin isotype in the DBA/2 response. CONCLUSION: Our study demonstrates that Mc cells can produce "autoantibodies" and points to a role of Mc in the biology of autoimmune diseases, including RA.

The skin sensitization adverse outcome pathway: exploring the role of mechanistic understanding for higher tier risk assessment.

For over a decade, the skin sensitization Adverse Outcome Pathway (AOP) has served as a useful framework for development of novel in chemico and in vitro assays for use in skin sensitization hazard and risk assessment. Since its establishment, the AOP framework further fueled the existing efforts in new assay development and stimulated a plethora of activities with particular focus on validation, reproducibility and interpretation of individual assays and combination of assay outputs for use in hazard/risk assessment. In parallel, research efforts have also accelerated in pace, providing new molecular and dynamic insight into key events leading to sensitization. In light of novel hypotheses emerging from over a decade of focused research effort, mechanistic evidence relating to the key events in the skin sensitization AOP may complement the tools currently used in risk assessment. We reviewed the recent advances unraveling the complexity of molecular events in sensitization and signpost the most promising avenues for further exploration and development of useful assays.

A methamphetamine vaccine using short monoamine and diamine peptide linkers and poly-mannose.

Methamphetamine (METH) substance use disorder is a long-standing and ever-growing public health concern. Efforts to develop successful immunotherapies are ongoing with vaccines that generate strong antibody responses are an area of significant research interest. Herein, we describe the development of a METH Hapten conjugate vaccine comprised of either two short-length peptides as linkers and mannan as an immunogenic delivery carrier. Initially, Hapten 1 (with a monoamine linker) and Hapten 2 (with a diamine linker) were synthesised. Each step of the Hapten synthesis were characterized by LC-MS and purified by Flash Chromatography and the identity of the purified Haptens were confirmed by 1H NMR. Haptens were conjugated with mannan (a polymannose), and conjugation efficiency was confirmed by LC-MS, TLC, 1H NMR, and 2,4 DNPH tests. The immunogenic potential of the two conjugated vaccines were assessed in mice with a 3-dose regimen. Concentrations of anti-METH antibodies were measured by enzyme-linked immunosorbent assay. All the analytical techniques confirmed the identity of Hapten 1 and 2 during the synthetic phase. Similarly, all the analytical approaches confirmed the conjugation between the Haptens and mannan. Mouse immunogenicity studies confirmed that both vaccine candidates were immunogenic and the vaccine with the monoamine linker plus adjuvants induced the highest antibody response after the second booster.

Rapid immunochemical methods for the analysis of proquinazid in strawberry QuEChERS extracts.

Proquinazid is a new-generation fungicide authorized in the EU for combating powdery mildew infections in high-value crops. Due to the perishable nature of fruits, alternative analytical methods are necessary to protect consumer's health from pesticide residues. Currently, immunoassays are a well-established approach for rapidly monitoring chemical contaminants. However, the production of high-quality immunoreagents, such as antibodies and bioconjugates, is essential. This study presents a newly designed hapten that maintains the characteristic moieties of proquinazid unmodified. The linear aliphatic substituents of this molecule were used to introduce the spacer arm. A three-step synthesis strategy was optimized to prepare a hapten that displays the entire 6-iodoquinazolin-4(3H)-one moiety with excellent yields. The N-hydroxysuccimidyl ester of the hapten was activated and purified to prepare a protein conjugate with high hapten density, which was used as an immunogen. Antibodies were raised and competitive enzyme-linked immunosorbent assays were developed. To enhance the assay's sensitivity, two additional heterologous haptens were prepared by modifying the halogenated substituent at C-6. The optimized assays demonstrated low limits of detection in buffer, approximately 0.05 µg/L. When applied to the analysis of proquinazid in QuEChERS extracts of strawberry samples, the immunoassays produced precise and accurate results, particularly in the 10-1000 µg/kg range.

Establishment of a Quenchbody-based L-thyroxine detection method and its comparison with ELISA systems.

The quantification of L-thyroxine (T4) is crucial for regulating metabolism, diagnosing diseases, and monitoring the efficacy of T4 replacement therapy. However, because T4 is a hapten biomarker with a molecular weight of 777 g/mol, conventional immunoassay approaches, including Western blotting and some types of ELISA, have limited accuracy in the quantification of small molecules, including T4. Furthermore, these methods are time-consuming and involve multiple incubation and reaction steps. Therefore, a novel immunoassay method is required for simple and rapid on-site detection of T4. In this study, we expressed a recombinant anti-T4 single-chain variable fragment (scFv) in soluble form using Escherichia coli. The scFv exhibited high T4-binding efficiency, and T4 concentration-dependent titration curves indicated that the sandwich ELISA could detect T4 in the nanogram range. We labeled the scFv using a fluorescent dye for a Quenchbody (Q-body)-based one-pot immunoassay, which yielded a T4 concentration-dependent fluorescent response in 3 min. A comparison of the Q-body-based T4 detection system with ELISA-based methods demonstrated that the ELISA system was more sensitive but the Q-body assay was more rapid. Therefore, both ELISA and Q-body systems can be used depending on the experimental purpose, with the newly developed anti-T4 Q-body system being applicable for convenient in situ immunoassay of T4.

Chemical tools for the opioids.

The opioids are potent and widely used pain management medicines despite also possessing severe liabilities that have fueled the opioid crisis. The pharmacological properties of the opioids primarily derive from agonism or antagonism of the opioid receptors, but additional effects may arise from specific compounds, opioid receptors, or independent targets. The study of the opioids, their receptors, and the development of remediation strategies has benefitted from derivatization of the opioids as chemical tools. While these studies have primarily focused on the opioids in the context of the opioid receptors, these chemical tools may also play a role in delineating mechanisms that are independent of the opioid receptors. In this review, we describe recent advances in the development and applications of opioid derivatives as chemical tools and highlight opportunities for the future.

Opioid-Based Haptens: Development of Immunotherapy.

Over the past decades, extensive preclinical research has been conducted to develop vaccinations to protect against substance use disorder caused by opioids, nicotine, cocaine, and designer drugs. Morphine or fentanyl derivatives are small molecules, and these compounds are not immunogenic, but when conjugated as haptens to a carrier protein will elicit the production of antibodies capable of reacting specifically with the unconjugated hapten or its parent compound. The position of the attachment in opioid haptens to the carrier protein will influence the specificity of the antiserum produced in immunized animals with the hapten-carrier conjugate. Immunoassays for the determination of opioid drugs are based on the ability of drugs to inhibit the reaction between drug-specific antibodies and the corresponding drug-carrier conjugate or the corresponding labelled hapten. Pharmacological studies of the hapten-carrier conjugates resulted in the development of vaccines for treating opioid use disorders (OUDs). Immunotherapy for opioid addiction includes the induction of anti-drug vaccines which are composed of a hapten, a carrier protein, and adjuvants. In this review we survey the design of opioid haptens, the development of the opioid radioimmunoassay, and the results of immunotherapy for OUDs.

Structural insight to elucidate the binding specificity of the anti-cortisol Fab fragment with glucocorticoids.

Cortisol is a steroid hormone that is produced by the adrenal gland. It is a primary stress hormone that increases glucose levels in the blood stream. High concentrations of cortisol in the body can be used as a biomarker for acute and chronic stress and related mental and physiological disorders. Therefore, the accurate quantification of cortisol levels in body fluids is essential for clinical diagnosis. In this article, we describe the isolation of recombinant anti-cortisol antibodies with high affinity for cortisol and discover their cross-reactivity with other glucocorticoids. To describe the cortisol binding site and elucidate the structural basis for the binding specificity, the high-resolution crystal structures of the anti-cortisol (17) Fab fragment in the absence of glucocorticoid (2.00 Å) and the presence of cortisol (2.26 Å), corticosterone (1.86 Å), cortisone (1.85 Å) and prednisolone (2.00 Å) were determined. To our knowledge, this is the first determined crystal structure of a cortisol-specific antibody. The recognition of cortisol is driven by hydrophobic interactions and hydrogen bonding at the protein-ligand interface coupled with a conformational transition. Comparison of ligand-free and ligand-bound structures showed that the side chains of residues Tyr58-H and Arg56-H can undergo local conformational changes at the binding site, most likely prior to the binding event via a conformational selection mechanism. Compared to other anti-steroid antibody-antigen complexes, (17) Fab possesses a structurally unique steroid binding site, as the H3 loop from the CDR area has only a minor contribution, but framework residues have a prominent contribution to hapten binding.

Viral infections and drug hypersensitivity.

Virus infections and T-cell-mediated drug hypersensitivity reactions (DHR) can influence each other. In most instances, systemic virus infections appear first. They may prime the reactivity to drugs in two ways: First, by virus-induced second signals: certain drugs like ß-lactam antibiotics are haptens and covalently bind to various soluble and tissue proteins, thereby forming novel antigens. Under homeostatic conditions, these neo-antigens do not induce an immune reaction, probably because co-stimulation is missing. During a virus infection, the hapten-modified peptides are presented in an immune-stimulatory environment with co-stimulation. A drug-specific immune reaction may develop and manifest as exanthema. Second, by increased pharmacological interactions with immune receptors (p-i): drugs tend to bind to proteins and may even bind to immune receptors. Without viral infections, this low affine binding may be insufficient to elicit T-cell activation. During a viral infection, immune receptors are more abundantly expressed and allow more interactions to occur. This increases the overall avidity of p-i reactions and may even be sufficient for T-cell activation and symptoms. There is a situation where the virus-DHR sequence of events is inversed: in drug reaction with eosinophilia and systemic symptoms (DRESS), a severe DHR can precede reactivation and viremia of various herpes viruses. One could explain this phenomenon by the massive p-i mediated immune stimulation during acute DRESS, which coincidentally activates many herpes virus-specific T cells. Through p-i stimulation, they develop a cytotoxic activity by killing herpes peptide-expressing cells and releasing herpes viruses. These concepts could explain the often transient nature of DHR occurring during viral infections and the often asymptomatic herpes-virus viraemia after DRESS.

Propionic Acid Groups and Multiple Aromatic Rings Induce Binding of Ketoprofen and Naproxen to the Hydrophobic Core of Bovine Serum Albumin.

Ketoprofen (KP), which causes photosensitivity by interacting with serum albumin (SA), and three drugs, ibuprofen (IBP), naproxen (NPX), and diazepam (DZP), which share the same binding site, were investigated for their interaction with bovine SA (BSA). For KP, DZP, and IBP, where drug-concentration-dependent quenching of BSA-intrinsic fluorescence was observed, a modified Stern-Volmer plot showed that dynamic quenching was dominant for KP and static quenching was dominant for DZP and IBP. However, this alone cannot be compared with NPX. Therefore, by performing singular value decomposition (SVD) fluorescence spectroscopy, we were able to find the behavior of the drug-concentration-dependent Langmuir-type principal component vectors. KSVD obtained by the Langmuir equation showed a high correlation with the static extinction constant V. Therefore, KSVD indicates the association constant of the drug with BSA and it was found that NPX and IBP had higher values than KP. Finally, in the analysis of the temperature factors of amino acid residues in each drug-binding region and Trp residues, KP and NPX significantly reduced these temperature factors whereas DZP and IBP hardly changed them. This result is consistent with the dynamic and static quenching dominance in the total quenching mechanism. Summarizing the results so far, it was shown that penetration into the hydrophobic core inside BSA can be achieved not only by one of the multiple aromatic rings and propionic acid groups but also by the joint effect of both. In this study, SVD enabled us to extract information on drug adsorption to BSA from fluorescence spectra. Furthermore, the application of SVD is expected to make it possible to perform fluorescence analysis for drug binding to proteins without being limited by the fluorescence properties of the drug.

Natural killer cell-mediated contact dermatitis-like reaction induced by treatment with TLR3 ligand poly(I:C).

INTRODUCTION: Poly(I:C) is recognised by endosomal Toll-like receptor 3 (TLR3) and activates cytotoxic CD8(+) lymphocytes and natural killer (NK) cells. It has been shown that the viral TLR3 agonist induces robust and long-lasting T-cell-mediated responses. In addition, TLR3 modulates the contact hypersensitivity reaction. OBJECTIVE: This study aimed to determine whether poly(I:C) injection can induce NK-mediated hapten reactivity in mice. METHODS: Mice were treated with poly(I:C), and their response to dinitrofluorobenzene hapten was measured by assessing ear swelling and serum interferon gamma (IFN-γ) production. Adoptive cell transfer and cell sorting were used to investigate the mechanism of the reaction, and the phenotype of poly(I:C)-activated liver NK cells was determined by flow cytometry analysis. RESULTS: The results showed that poly(I:C) administration increased ear swelling, serum IFN-γ levels and the response to hapten in both immunocompetent and T- and B-cell-deficient mice. Only liver poly(I:C)-activated DX5(+) NK cells were able to transfer reactivity to hapten into a naive recipient. Induction of liver NK cells after poly(I:C) administration was TLR3/TRIF- and IFN-γ-dependent, interleukin 12-independent, and not modulated by MyD88. CONCLUSION: This study provides new insights into how poly(I:C) stimulates NK-mediated reactivity to hapten and suggests that liver NK cells may modulate the immune response to non-pathogenic factors during viral infection.

The European baseline series and recommended additions: 2023.

The European baseline series was last updated in 2019. This article discusses the reasoning behind a further iteration of the series for 2023.

Production of monoclonal antibody against tylosin and tilmicosin with homogeneous cross-reactivity and its application in lateral flow immunoassay.

To avoid false negative results due to the low cross-reactivity rate (CR) in rapid immunoassay, a group-specific antibody with homogeneous CR toward target compounds is needed for accuracy. In this study, tylosin (TYL) and tilmicosin (TM) were selected as model molecules. Firstly, two-dimensional similarity, electrostatic potential energy, spatial conformation and charge distribution of the haptens TYL-CMO, TYL-6-ACA, TYL-4-APA, TYL-CHO and DES-CMO and target compounds of TYL and TM were obtained using Gaussian 09W and Discovery Studio. The optimal hapten was DES-CMO because it is the most similar to TYL and TM. Subsequently, the mAb 14D5 cell line was obtained with IC50 values of 1.59 and 1.72 ng/mL for TYL and TM, respectively, and a CR of 92.44%. Finally, amorphous carbon nanoparticles (ACNPs) were conjugated with mAb 14D5 to develop an accurate lateral flow immunoassay (LFA) for detection of TYL and TM by the reflectance value under natural light. The recoveries of TYL and TM ranged from 77.18 to 112.04% with coefficient of variation < 13.43%. The cut-off value in milk samples was 8 ng/mL, and the limits of detection were 11.44, 15.96, 22.29 and 25.53 µg/kg for chicken muscle, bovine muscle, porcine muscle and porcine liver samples, respectively, and the results being consistent with HPLC-UV. The results suggest that the developed LFA is accurate and potentially useful for on-site screening of TYL and TM in milk and animal tissue samples.

Skin codelivery of contact sensitizers and neurokinin-1 receptor antagonists integrated in microneedle arrays suppresses allergic contact dermatitis.

BACKGROUND: Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type 1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P and hemokinin 1, which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. OBJECTIVE: We sought to develop an Ag-specific immunosuppressive approach to treat CD by skin codelivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). METHODS: In vivo mouse models of contact hypersensitivity and ex vivo models of human skin were used to delineate the effects and mechanisms of NK1R signaling and the immunosuppressive effects of the contact sensitizer NK1R antagonist MNA in CD. RESULTS: We demonstrated in mice that CD requires NK1R signaling by substance P and hemokinin 1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells, prevented CD. Skin codelivery of hapten or Ag MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells, and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. CONCLUSIONS: Immunoregulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD are caused by type 1 immunity.

Role of Innate Immunity in Allergic Contact Dermatitis: An Update.

Our understanding of allergic contact dermatitis mechanisms has progressed over the past decade. Innate immune cells that are involved in the pathogenesis of allergic contact dermatitis include Langerhans cells, dermal dendritic cells, macrophages, mast cells, innate lymphoid cells (ILCs), neutrophils, eosinophils, and basophils. ILCs can be subcategorized as group 1 (natural killer cells; ILC1) in association with Th1, group 2 (ILC2) in association with Th2, and group 3 (lymphoid tissue-inducer cells; ILC3) in association with Th17. Pattern recognition receptors (PRRs) including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) in innate immune cells recognize damage-associated molecular patterns (DAMPs) and cascade the signal to produce several cytokines and chemokines including tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-γ, interleukin (IL)-1ß, IL-4, IL-6, IL-12, IL-13, IL-17, IL-18, and IL-23. Here we discuss the recent findings showing the roles of the innate immune system in allergic contact dermatitis during the sensitization and elicitation phases.

Antibody-dependent cellular phagocytosis of tropomyosin receptor kinase C (TrkC) expressing cancer cells for targeted immunotherapy.

Conventional cancer therapies such as chemotherapy are non-selective and induce immune system anergy, which lead to serious side effects and tumor relapse. It is a challenge to prime the body's immune system in the cancer-bearing subject to produce cancer antigen-targeting antibodies, as most tumor-associated antigens are expressed abundantly in cancer cells and some of normal cells. This study illustrates how hapten-based pre-immunization (for anti-hapten antibodies production) combined with cancer receptor labeling with hapten antigen constructs can elicit antibody-dependent cellular phagocytosis (ADCP). Thus, the hapten antigen 2,4-dinitrophenol (DNP) was covalently combined with a cancer receptor-binding dipeptide (IYIY) to form a dipeptide-hapten construct (IYIY-DNP, MW = 1322.33) that targets the tropomyosin receptor kinase C (TrkC)-expressed on the surface of metastatic cancer cells. IYIY-DNP facilitated selective association of RAW264.7 macrophages to the TrkC expressing 4T1 cancer cells in vitro, forming cell aggregates in the presence of anti-DNP antibodies, suggesting initiation of anti-DNP antibody-dependent cancer cell recognition of macrophages by the IYIY-DNP. In in vivo, IYIY-DNP at 10 mg/kg suppressed growth of 4T1 tumors in DNP-immunized BALB/c mice by 45% (p < 0.05), when comparing the area under the tumor growth curve to that of the saline-treated DNP-immunized mice. Meanwhile, IYIY-DNP at 10 mg/kg had no effect on TrkC-negative 67NR tumor-bearing mice immunized with DNP. Tumor growth suppression activity of IYIY-DNP in DNP-immunized mice was associated with an increase in the anti-DNP IgG (7.3 × 106 ± 1.6 U/mL) and IgM (0.9 × 106 ± 0.07 U/mL) antibodies after five cycles of DNP treatment, demonstrated potential for hapten-based pre-immunization then treatment with IYIY-DNP to elicit ADCP for improved immunotherapy of TrkC expressing cancers.

Epicutaneous Patch Testing in Type IV Allergy Diagnostics: State of the Art and Best Practice Recommendations.

This chapter summarises all relevant aspects of patch testing, closely following recommendations outlined in a recent European, and a German S3 guideline on diagnostic patch testing with contact allergens and medicinal products (drugs). Patch testing is indicated in patients suspected of suffering, or having been suffering, from delayed-type hypersensitivity leading to allergic contact dermatitis or other skin and mucosal diseases. Sections of this chapter include detailed indications, reasons for possibly postponing the test, considerations on choosing haptens (contact allergens) to test, various aspects of the application of patch test allergen preparations (storage, dosing) and of testing with individual materials provided by the patients. Special aspects of patch testing in cutaneous adverse drug reactions, children, or occupational contact dermatitis are outlined. Supplemental test methods, notably the repeated open application test, are briefly described. Finally, the final evaluation in terms of assessment of clinical relevance of reactions and patient counselling are outlined.

Design, Synthesis, and In Vivo Evaluation of C1-Linked 4,5-Epoxymorphinan Haptens for Heroin Vaccines.

In our continuing effort to develop effective anti-heroin vaccines as potential medications for the treatment of opioid use disorder, herein we present the design and synthesis of the haptens: 1-AmidoMorHap (1), 1-AmidoMorHap epimer (2), 1 Amido-DihydroMorHap (3), and 1 Amido-DihydroMorHap epimer (4). This is the first report of hydrolytically stable haptenic surrogates of heroin with the attachment site at the C1 position in the 4,5-epoxymorophinan nucleus. We prepared respective tetanus toxoid (TT)-hapten conjugates as heroin vaccine immunogens and evaluated their efficacy in vivo. We showed that all TT-hapten conjugates induced high antibody endpoint titers against the targets but only haptens 2 and 3 can induce protective effects against heroin in vivo. The epimeric analogues of these haptens, 1 and 4, failed to protect mice from the effects of heroin. We also showed that the in vivo efficacy is consistent with the results of the in vitro drug sequestration assay. Attachment of the linker at the C1 position induced antibodies with weak binding to the target drugs. Only TT-2 and TT-3 yielded antibodies that bound heroin and 6-acetyl morphine. None of the TT-hapten conjugates induced antibodies that cross-reacted with morphine, methadone, naloxone, or naltrexone, and only TT-3 interacted weakly with buprenorphine, and that subtle structural difference, especially at the C6 position, can vastly alter the specificity of the induced antibodies. This study is an important contribution in the field of vaccine development against small-molecule targets, providing proof that the chirality at C6 in these epoxymorphinans is a vital key to their effectiveness.