Independent infections of porcine deltacoronavirus among Haitian children.

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3-5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human-animal contact.

Molecular Mechanisms of Lupus Susceptibility Allele PBX1D.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Sigma Receptor Ligands Prevent COVID Mortality In Vivo: Implications for Future Therapeutics.

The emergence of lethal coronaviruses follows a periodic pattern which suggests a recurring cycle of outbreaks. It remains uncertain as to when the next lethal coronavirus will emerge, though its eventual emergence appears to be inevitable. New mutations in evolving SARS-CoV-2 variants have provided resistance to current antiviral drugs, monoclonal antibodies, and vaccines, reducing their therapeutic efficacy. This underscores the urgent need to investigate alternative therapeutic approaches. Sigma receptors have been unexpectedly linked to the SARS-CoV-2 life cycle due to the direct antiviral effect of their ligands. Coronavirus-induced cell stress facilitates the formation of an ER-derived complex conducive to its replication. Sigma receptor ligands are believed to prevent the formation of this complex. Repurposing FDA-approved drugs for COVID-19 offers a timely and cost-efficient strategy to find treatments with established safety profiles. Notably, diphenhydramine, a sigma receptor ligand, is thought to counteract the virus by inhibiting the creation of ER-derived replication vesicles. Furthermore, lactoferrin, a well-characterized immunomodulatory protein, has shown antiviral efficacy against SARS-CoV-2 both in laboratory settings and in living organisms. In the present study, we aimed to explore the impact of sigma receptor ligands on SARS-CoV-2-induced mortality in ACE2-transgenic mice. We assessed the effects of an investigational antiviral drug combination comprising a sigma receptor ligand and an immunomodulatory protein. Mice treated with sigma-2 receptor ligands or diphenhydramine and lactoferrin exhibited improved survival rates and rapid rebound in mass following the SARS-CoV-2 challenge compared to mock-treated animals. Clinical translation of these findings may support the discovery of new treatment and research strategies for SARS-CoV-2.

Emerging mutation patterns in SARS-CoV-2 variants.

There is an urgent need to understand the functional effects of mutations in emerging variants of SARS-CoV-2. Variants of concern (alpha, beta, gamma and delta) acquired four patterns of spike glycoprotein mutations that enhance transmissibility and immune evasion: 1) mutations in the N-terminal domain (NTD), 2) mutations in the Receptor Binding Domain (RBD), 3) mutations at interchain contacts of the spike trimer, and 4) furin cleavage site mutations. Most distinguishing mutations among variants of concern are exhibited in the NTD, localized to sites of high structural flexibility. Emerging variants of interest such as mu, lambda and C.1.2 exhibit the same patterns of mutations as variants of concern. There is a strong likelihood that SARS-CoV-2 variants will continue to emerge with mutations in these defined patterns, thus providing a basis for the development of next line antiviral drugs and vaccine candidates.

2.1 Å crystal structure of the Mycobacterium tuberculosis serine hydrolase, Hip1, in its anhydro-form (Anhydrohip1).

The 2.6 Å crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M. tuberculosis (Mtb), serine hydrolase drug target. To begin mapping the active site of Hip1, we cocrystallized Hip1 with the irreversible serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). We chose AEBSF for cocrystallization with Hip1 since the similar inhibitor, phenylmethylsulfonyl fluoride (PMSF), interestingly exhibited no activity against Hip1. We obtained crystals that diffracted to 2.1 Å but to our bewilderment, we did not observe any electron density for the inhibitor in the omit map for the Hip1-AEBSF complex. Rather, in the active site, dehydroalanine (dAla) was found to occupy the expected position of the catalytic Ser228, thus yielding anhydrohip1. Here we present a comparative analysis of the crystal structures of anhydrohip1 and Hip1 and provide a mechanism for the conversion of the enzyme to the anhydro-form through reaction with AEBSF. With the aid of molecular docking, we propose an explanation for the differential inhibition of Hip1 by AEBSF and PMSF. We also present a preliminary definition of the S1 and S2 pockets of the protease's active site and propose a mechanism for a ligand-induced conformational change within the S2 pocket. Finally, we expand upon the previous demarcation of the putative lipid binding pocket in the α-domain of the enzyme. We believe that this detailed analysis of the structures of anhydrohip1 and Hip1 provides valuable information useful for the structure-based drug design of novel Hip1-directed Mtb therapeutics.

Low-frequency variants in mildly symptomatic vaccine breakthrough infections presents a doubled-edged sword.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) has raised questions regarding vaccine protection against SARS-CoV-2 infection, transmission, and ongoing virus evolution. Twenty-three mildly symptomatic "vaccination breakthrough" infections were identified as early as January 2021 in Alachua County, Florida, among individuals fully vaccinated with either the BNT162b2 (Pfizer) or the Ad26 (Janssen/J&J) vaccines. SARS-CoV-2 genomes were successfully generated for 11 of the vaccine breakthroughs, and 878 individuals in the surrounding area and were included for reference-based phylogenetic investigation. These 11 individuals were characterized by infection with VOCs, but also low-frequency variants present within the surrounding population. Low-frequency mutations were observed, which have been more recently identified as mutations of interest owing to their location within targeted immune epitopes (P812L) and association with increased replicative capacity (L18F). We present these results to posit the nature of the efficacy of vaccines in reducing symptoms as both a blessing and a curse-as vaccination becomes more widespread and self-motivated testing reduced owing to the absence of severe symptoms, we face the challenge of early recognition of novel mutations of potential concern. This case study highlights the critical need for continued testing and monitoring of infection and transmission among individuals regardless of vaccination status.

Human Leukocyte Antigen B*14:01 and B*35:01 Are Associated With Trimethoprim-Sulfamethoxazole Induced Liver Injury.

BACKGROUND AND AIMS: Trimethoprim (TMP)-sulfamethoxazole (SMX) is an important cause of idiosyncratic drug-induced liver injury (DILI), but its genetic risk factors are not well understood. This study investigated the relationship between variants in the human leukocyte antigen (HLA) class 1 and 2 genes and well-characterized cases of TMP-SMX DILI. APPROACH AND RESULTS: European American and African American persons with TMP-SMX DILI were compared with respective population controls. HLA sequencing was performed by Illumina MiSeq (Illumina, San Diego, CA) for cases. The HLA genotype imputation with attribute bagging program was used to impute HLA alleles for controls. The allele frequency difference between case patients and controls was tested by Fisher's exact tests for each ethnic group. For European Americans, multivariable logistic regression with Firth penalization was used to test the HLA allelic effect after adjusting for age and the top two principal components. Molecular docking was performed to assess HLA binding with TMP and SMX. The European American subset had 51 case patients and 12,156 controls, whereas the African American subset had 10 case patients and 5,439 controls. Four HLA alleles were significantly associated in the European American subset, with HLA-B*14:01 ranking at the top (odds ratio, 9.20; 95% confidence interval, 3.16, 22.35; P = 0.0003) after covariate adjustment. All carriers of HLA-B*14:01 with TMP-SMX DILI possessed HLA-C*08:02, another significant allele (P = 0.0026). This pattern was supported by HLA-B*14:01-HLA-C*08:02 haplotype association (P = 1.33 × 10-5 ). For the African American patients, HLA-B*35:01 had 2.8-fold higher frequency in case patients than in controls, with 5 of 10 patients carrying this allele. Molecular docking showed cysteine at position 67 in HLA-B*14:01 and phenylalanine at position 67 in HLA-B*35:01 to be the predictive binding sites for SMX metabolites. CONCLUSIONS: HLA-B*14:01 is associated with TMP-SMX DILI in European Americans, and HLA-B*35:01 may be a potential genetic risk factor for African Americans.

Identification of antiviral antihistamines for COVID-19 repurposing.

There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Although repurposed drugs with favorable safety profiles could have significant benefit, widely available prevention or treatment options for COVID-19 have yet to be identified. Efforts to identify approved drugs with in vitro activity against SARS-CoV-2 resulted in identification of antiviral sigma-1 receptor ligands, including antihistamines in the histamine-1 receptor binding class. We identified antihistamine candidates for repurposing by mining electronic health records of usage in population of more than 219,000 subjects tested for SARS-CoV-2. Usage of diphenhydramine, hydroxyzine and azelastine was associated with reduced incidence of SARS-CoV-2 positivity in subjects greater than age 61. We found diphenhydramine, hydroxyzine and azelastine to exhibit direct antiviral activity against SARS-CoV-2 in vitro. Although mechanisms by which specific antihistamines exert antiviral effects is not clear, hydroxyzine, and possibly azelastine, bind Angiotensin Converting Enzyme-2 (ACE2) and the sigma-1 receptor as off-targets. Clinical studies are needed to measure the effectiveness of diphenhydramine, hydroxyzine and azelastine for disease prevention, for early intervention, or as adjuvant therapy for severe COVID-19.

Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of Neurolysin.

Neurolysin (Nln) is a recently recognized endogenous mechanism functioning to preserve the brain from ischemic injury. To further understand the pathophysiological function of this peptidase in stroke and other neurologic disorders, the present study was designed to identify small molecule activators of Nln. Using a computational approach, the structure of Nln was explored, which was followed by docking and in silico screening of ∼140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database. Top ranking compounds were evaluated in an Nln enzymatic assay, and two hit histidine-dipeptides were further studied in detail. The identified dipeptides enhanced the rate of synthetic substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Nln in a concentration-dependent manner (micromolar A50 and Amax ≥ 300%) but had negligible effect on activity of closely related peptidases. Both dipeptides also enhanced hydrolysis of Nln endogenous substrates neurotensin, angiotensin I, and bradykinin and increased efficiency of the synthetic substrate hydrolysis (Vmax/Km ratio) in a concentration-dependent manner. The dipeptides and competitive inhibitor dynorphin A (1-13) did not affect each other's affinity for Nln, suggesting differing nature of their respective binding sites. Lastly, drug affinity responsive target stability (DARTS) and differential scanning fluorimetry (DSF) assays confirmed concentration-dependent interaction of Nln with the activator molecule. This is the first study demonstrating that Nln activity can be enhanced by small molecules, although the peptidic nature and low potency of the activators limit their application. The identified dipeptides provide a chemical scaffold to develop high-potency, drug-like molecules as research tools and potential drug leads. SIGNIFICANCE STATEMENT: This study describes discovery of two molecules that selectively enhance activity of peptidase Nln-a newly recognized cerebroprotective mechanism in the poststroke brain. The identified molecules will serve as a chemical scaffold for development of drug-like molecules to further study Nln and may become lead structures for a new class of drugs. In addition, our conceptual and methodological framework and research findings might be used for other peptidases and enzymes, the activation of which bears therapeutic potential.

Sweet-Tasting Ionic Conjugates of Local Anesthetics and Vasoconstrictors.

Local anesthetics are widely utilized in dentistry, cosmetology, and medicine. Local anesthesia is essential to providing a pain-free experience during dental and local surgeries as well as cosmetic procedures. However, the injection itself may produce discomfort and be a source of aversion. A novel approach toward the taste modulation of local anesthetics is proposed, in which the anesthetics of the "-caine" family serve as cations and are coupled with anionic sweeteners such as saccharinate and acesulfamate. Ionic conjugates of vasoconstrictor epinephrine such as epinephrine saccharinate and epinephrine acesulfamate have also been synthesized. Novel ionic conjugates were developed using anion exchange techniques. Reported compounds are sweet-tasting and are safe to use both topically and as injections.

Identification of small molecules by screening a mixture-based scaffold compound library for treatment of alpha-1 antitrypsin deficiency.

This study aimed to identify small molecules that have the potential to treat alpha1-antitrypsin deficiency (AATD) by screening compounds available from a mixture-based scaffold library. 93 scaffold libraries (total diversity of >30 million compounds in mixture format) were screened using a cell model of AATD in order to identify samples that could either reduce intracellular aggregation of Z-form AAT protein, increase extracellular secretion of Z-AAT or both. Mixture libraries containing compounds with in vitro activity, for example library 1295, were screened further to identify individual active compounds. The mixture format of the scaffold library allowed for some preliminary structure-activity relationships to be developed and also enabled the rapid selection of a promising scaffold. Utilizing this scaffold, 1295, a collection of individual "control" compounds contained in the 1295 mixture sample were then screened. A sub-library of individual "control" compounds featuring structural diversity at position R1 (1295.R1), was screened and 7 compounds were found to reduce the intracellular accumulation of Z-AAT without affecting cell viability at a concentration of 25ug/ml (about 50 µM). Screening sub-libraries featuring structural diversity at R2 and R3 (1295.R2 and 1295.R3) identified an additional 15 active compounds. Titration experiments identified 3 compounds from the 1295.R2 library that retained activity at 5ug/ml (approx. 10uM). One compound (1295.263) from 1295.R2 decreased intracellular levels of Z-AAT without affecting cell viability and wild-type AAT levels at the concentration of 5ug/ml. Molecular docking of this compound to the Z-AAT crystal structure identified a potential binding site near the C-terminal domain, an identified polymerization site. Our results indicate that screening large mixture-based compound libraries can be used to identify small molecules that may have the potential to treat AATD and other disease.

HLA-A*32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms.

BACKGROUND: Vancomycin is a prevalent cause of the severe hypersensitivity syndrome drug reaction with eosinophilia and systemic symptoms (DRESS), which leads to significant morbidity and mortality and commonly occurs in the setting of combination antibiotic therapy, affecting future treatment choices. Variations in HLA class I in particular have been associated with serious T cell-mediated adverse drug reactions, which has led to preventive screening strategies for some drugs. OBJECTIVE: We sought to determine whether variation in the HLA region is associated with vancomycin-induced DRESS. METHODS: Probable vancomycin-induced DRESS cases were matched 1:2 with tolerant control subjects based on sex, race, and age by using BioVU, Vanderbilt's deidentified electronic health record database. Associations between DRESS and carriage of HLA class I and II alleles were assessed by means of conditional logistic regression. An extended sample set from BioVU was used to conduct a time-to-event analysis of those exposed to vancomycin with and without the identified HLA risk allele. RESULTS: Twenty-three subjects met the inclusion criteria for vancomycin-associated DRESS. Nineteen (82.6%) of 23 cases carried HLA-A*32:01 compared with 0 (0%) of 46 of the matched vancomycin-tolerant control subjects (P = 1 × 10-8) and 6.3% of the BioVU population (n = 54,249, P = 2 × 10-16). Time-to-event analysis of DRESS development during vancomycin treatment among the HLA-A*32:01-positive group indicated that 19.2% had DRESS and did so within 4 weeks. CONCLUSIONS: HLA-A*32:01 is strongly associated with vancomycin-induced DRESS in a population of predominantly European ancestry. HLA-A*32:01 testing could improve antibiotic safety, help implicate vancomycin as the causal drug, and preserve future treatment options with coadministered antibiotics.

Multiple Recombination Events and Strong Purifying Selection at the Origin of SARS-CoV-2 Spike Glycoprotein Increased Correlated Dynamic Movements.

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.

Dapsone- and nitroso dapsone-specific activation of T cells from hypersensitive patients expressing the risk allele HLA-B*13:01.

BACKGROUND: Research into drug hypersensitivity associated with the expression of specific HLA alleles has focussed on the interaction between parent drug and the HLA with no attention given to reactive metabolites. For this reason, we have studied HLA-B*13:01-linked dapsone hypersensitivity to (a) explore whether the parent drug and/or nitroso metabolite activate T cells and (b) determine whether HLA-B*13:01 is involved in the response. METHODS: Peripheral blood mononuclear cells (PBMC) from six patients were cultured with dapsone and nitroso dapsone, and proliferative responses and IFN-γ release were measured. Dapsone- and nitroso dapsone-specific T-cell clones were generated and phenotype, function, HLA allele restriction, and cross-reactivity assessed. Dapsone intermediates were characterized by mass spectrometry. RESULTS: Peripheral blood mononuclear cells from six patients and cloned T cells proliferated and secreted Th1/2/22 cytokines when stimulated with dapsone (clones: n = 395; 80% CD4+ CXCR3hi CCR4hi , 20% CD8+CXCR3hi CCR4hi CCR6hi CCR9hi CCR10hi ) and nitroso dapsone (clones: n = 399; 78% CD4+, 22% CD8+ with same chemokine receptor profile). CD4+ and CD8+ clones were HLA class II and class I restricted, respectively, and displayed three patterns of reactivity: compound specific, weakly cross-reactive, and strongly cross-reactive. Nitroso dapsone formed dimers in culture and was reduced to dapsone, providing a rationale for the cross-reactivity. T-cell responses to nitroso dapsone were dependent on the formation of a cysteine-modified protein adduct, while dapsone interacted in a labile manner with antigen-presenting cells. CD8+ clones displayed an HLA-B*13:01-restricted pattern of activation. CONCLUSION: These studies describe the phenotype and function of dapsone- and nitroso dapsone-responsive CD4+ and CD8+ T cells from hypersensitive patients. Discovery of HLA-B*13:01-restricted CD8+ T-cell responses indicates that drugs and their reactive metabolites participate in HLA allele-linked forms of hypersensitivity.

Molecular docking predictions of fragrance binding to human leukocyte antigen molecules.

BACKGROUND: Over 4000 small chemicals have been identified as allergens capable of inducing skin sensitization. Many sensitizers are hypothesized to act as haptens producing novel antigens, which can be presented to T cells by human leukocyte antigens (HLAs). Recent studies suggest that some chemical allergens use hapten-independent mechanisms. OBJECTIVE: To determine whether molecular docking can identify HLA molecules that bind skin-sensitizing chemical allergens. METHODS: Structural models of HLA molecules were used as the basis for molecular docking of 22 chemical allergens. Allergens predicted to bind HLA-B*57:01 were tested for their ability to stimulate T cells by the use of proliferation and interferon-gamma enzyme-linked immunospot assays. RESULTS: Chemical allergens that did not satisfy the criteria for hapten activity in vitro were predicted to bind more strongly to common HLA isoforms than those with known hapten activity. HLA-B*57:01, which is an HLA allele required for drug hypersensitivity reactions, was predicted to bind several allergens, including benzyl benzoate, benzyl cinnamate, and benzyl salicylate. In in vitro T cell stimulation assays, benzyl salicylate and benzyl cinnamate were found to stimulate T cell responses from HLA-B*57:01 carriers. CONCLUSIONS: These data suggest that small-molecule skin sensitizers have the potential to interact with HLA, and show that T cell-based in vitro assays may be used to evaluate the immunogenicity of skin-sensitizing chemicals.

Association of the HLA-B*53:01 Allele With Drug Reaction With Eosinophilia and Systemic Symptoms (DRESS) Syndrome During Treatment of HIV Infection With Raltegravir.

Background: Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a rare, severe adverse event during treatment with raltegravir. The occurrence of DRESS syndrome during treatment with other drugs is strongly associated with particular HLA alleles. Methods: We performed HLA testing in 3 of the 5 patients previously reported to have developed raltegravir-induced DRESS syndrome and in 1 previously unreported patient. We then used virtual modeling to visualize interactions between raltegravir and the imputed HLA molecule. Results: Five of the 6 patients who developed raltegravir-induced DRESS syndrome were African, and 1 was Hispanic. HLA typing was performed in 4 patients, all of whom carried both the HLA-B*53 allele and the HLA-C*04 allele to which it is commonly haplotypic. No other HLA alleles were shared by all of the tested patients. Given the approximate prevalence of HLA-B*53 carriage in African (20%) and Hispanic (6%) populations, the probability of all 4 patients being HLA-B*53 carriers, and 2 of 3 African patients being homozygous for HLA-B*53:01, is approximately 0.00002. Conclusions: These data implicate the prevalent African allele HLA-B*53:01 in the immunopathogenesis of raltegravir-induced DRESS syndrome. Although the immunopathogenic mechanisms are currently unknown, virtual modeling suggests that raltegravir may bind within the antigen binding cleft of the HLA-B*53:01 molecule, but not within the closely related HLA-B*35:01 molecule. Further studies are necessary to confirm the strength of the association between carriage of the HLA-B*53:01 allele and raltegravir-induced DRESS syndrome, and the potential utility of HLA screening before raltegravir treatment.

A bony fish immunological receptor of the NITR multigene family mediates allogeneic recognition.

Novel immune-type receptors (NITRs) comprise an exceptionally large, diversified family of activating and inhibitory receptors that has been identified in bony fish. Here, we characterized the structure of an activating NITR that is expressed by a cytotoxic natural killer (NK)-like cell line and that specifically binds an allogeneic B cell target. A single amino acid residue within the NITR immunoglobulin variable (V)-type domain accounts for specificity of the interaction. Structures solved by X-ray crystallography revealed that the V-type domains of NITRs form homodimers resembling rearranging antigen-binding receptor heterodimers. CDR1 elements of both subunits of NITR dimers form ligand-binding surfaces that determine specificity for the nonself target. In the evolution of immune function, it appears that a specific NK type of innate recognition may be mediated by a complex germline multigene family of V structures resembling those that are somatically diversified in adaptive immunological responses.

Structural Elements Recognized by Abacavir-Induced T Cells.

Adverse drug reactions are one of the leading causes of morbidity and mortality in health care worldwide. Human leukocyte antigen (HLA) alleles have been strongly associated with drug hypersensitivities, and the causative drugs have been shown to stimulate specific T cells at the sites of autoimmune destruction. The structural elements recognized by drug-specific T cell receptors (TCRs) in vivo are poorly defined. Drug-stimulated T cells express TCRs specific for peptide/HLA complexes, but the characteristics of peptides (sequence, or endogenous or exogenous origin) presented in the context of small molecule drugs are not well studied. Using HLA-B*57:01 mediated hypersensitivity to abacavir as a model system, this study examines structural similarities of HLA presented peptides recognized by drug-specific TCRs. Using the crystal structure of HLA-B*57:01 complexed with abacavir and an immunogenic self peptide, VTTDIQVKV SPT5a 976-984, peptide side chains exhibiting flexibility and solvent exposure were identified as potential drug-specific T cell recognition motifs. Viral sequences with structural motifs similar to the immunogenic self peptide were identified. Abacavir-specific T cell clones were used to determine if virus peptides presented in the context of abacavir stimulate T cell responsiveness. An abacavir-specific T cell clone was stimulated by VTQQAQVRL, corresponding to HSV1/2 230-238, in the context of HLA-B*57:01. These data suggest the T cell polyclonal response to abacavir consists of multiple subsets, including T cells that recognize self peptide/HLA-B*57:01 complexes and crossreact with viral peptide/HLA-B*57:01 complexes due to similarity in TCR contact residues.