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Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida

Wagner B, Krebitz M, Buck D, Niggemann B, Yeang HY, Han KH, et al.

J Allergy Clin Immunol, 1999 Nov;104(5):1084-92.
PMID: 10550757

BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy.
OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.
METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.
RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.
CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.
Whole genome sequencing identifies genetic variants associated with co-trimoxazole hypersensitivity in Asians

Wang CW, Tassaneeyakul W, Chen CB, Chen WT, Teng YC, Huang CY, et al.

J Allergy Clin Immunol, 2021 04;147(4):1402-1412.
PMID: 32791162 DOI: 10.1016/j.jaci.2020.08.003

BACKGROUND: Co-trimoxazole, a sulfonamide antibiotic, is used to treat a variety of infections worldwide, and it remains a common first-line medicine for prophylaxis against Pneumocystis jiroveci pneumonia. However, it can cause severe cutaneous adverse reaction (SCAR), including Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia and systemic symptoms. The pathomechanism of co-trimoxazole-induced SCAR remains unclear.
OBJECTIVE: We aimed to investigate the genetic predisposition of co-trimoxazole-induced SCAR.
METHODS: We conducted a multicountry case-control association study that included 151 patients with of co-trimoxazole-induced SCAR and 4631 population controls from Taiwan, Thailand, and Malaysia, as well as 138 tolerant controls from Taiwan. Whole-genome sequencing was performed for the patients and population controls from Taiwan; it further validated the results from Thailand and Malaysia.
RESULTS: The whole-genome sequencing study (43 case patients vs 507 controls) discovered that the single-nucleotide polymorphism rs41554616, which is located between the HLA-B and MICA loci, had the strongest association with co-trimoxazole-induced SCAR (P = 8.2 × 10-9; odds ratio [OR] = 7.7). There were weak associations of variants in co-trimoxazole-related metabolizing enzymes (CYP2D6, GSTP1, GCLC, N-acetyltransferase [NAT2], and CYP2C8). A replication study using HLA genotyping revealed that HLA-B∗13:01 was strongly associated with co-trimoxazole-induced SCAR (the combined sample comprised 91 case patients vs 2545 controls [P = 7.2 × 10-21; OR = 8.7]). A strong HLA association was also observed in the case patients from Thailand (P = 3.2 × 10-5; OR = 3.6) and Malaysia (P = .002; OR = 12.8), respectively. A meta-analysis and phenotype stratification study further indicated a strong association between HLA-B∗13:01 and co-trimoxazole-induced drug reaction with eosinophilia and systemic symptoms (P = 4.2 × 10-23; OR = 40.1).
CONCLUSION: This study identified HLA-B∗13:01 as an important genetic factor associated with co-trimoxazole-induced SCAR in Asians.
Treg cell dysfunction and cutaneous exposure to S. aureus underlie eczema in DOCK8 deficiency

Wilkie H, Das M, Pelovitz T, Bainter W, Woods B, Alasharee M, et al.

J Allergy Clin Immunol, 2024 Jan 05.
PMID: 38185418 DOI: 10.1016/j.jaci.2023.12.020

BACKGROUND: DOCK8-deficient patients have severe eczema, elevated IgE and eosinophilia, features of Atopic Dermatitis (AD).
OBJECTIVE: To understand the mechanisms of eczema in DOCK8 deficiency.
METHODS: Skin biopsies were characterized for histology, immuno-fluorescence microscopy, and gene expression. Skin barrier function was measured by trans-epidermal water loss. Allergic skin inflammation was elicited in mice by epicutaneous (EC) sensitization with ovalbumin (OVA) or cutaneous application of S. aureus.
RESULTS: Skin lesions of DOCK8-deficient patients exhibited type-2 inflammation and the patients' skin was colonized by S. aureus, like in AD. Unlike in AD, DOCK8-deficient patients had a reduced FOXP3:CD4 ratio in their skin lesions, and their skin barrier function was intrinsically intact. Dock8-/- mice exhibited reduced numbers of cutaneous T regulatory cells (Tregs) and a normal skin barrier. Dock8-/- mice and mice with an inducible Dock8 deletion in Tregs exhibited increased allergic skin inflammation following EC sensitization with OVA. DOCK8 was shown to be important for Treg stability at sites of allergic inflammation and for the generation, survival, and suppressive activity of inducible Tregs (iTregs). Adoptive transfer of wild-type, but not DOCK8-deficient, OVA-specific iTregs suppressed allergic inflammation in OVA-sensitized skin of Dock8-/- mice. These mice developed severe allergic skin inflammation and elevated serum IgE levels following topical exposure to S. aureus. Both were attenuated following adoptive transfer of WT but not DOCK8 deficient Tregs CONCLUSION: Treg cell dysfunction increases susceptibility to allergic skin inflammation in DOCK8 deficiency and synergizes with cutaneous exposure to S. aureus to drive eczema in DOCK8 deficiency.
Hev b 5 and Hev b 13 as allergen markers to estimate the allergenic potency of latex gloves

Yeang HY, Arif SA, Raulf-Heimsoth M, Loke YH, Sander I, Sulong SH, et al.

J Allergy Clin Immunol, 2004 Sep;114(3):593-8.
PMID: 15356563 DOI: 10.1016/j.jaci.2004.05.039

BACKGROUND:
Sensitization to natural rubber latex has been linked to proteins from medical latex gloves. Various assays to estimate the amount of residual allergenic proteins extractable from latex gloves to assess their potential exposure hazard have inherent weaknesses.

OBJECTIVE:
This investigation was aimed at developing 2-site immunoenzymetric assays and identifying appropriate protein markers to assess the allergenic potential of latex gloves.

METHODS:
The presence of 6 latex allergens--Hev b 1, 2, 3, 5, 6, and 13--was measured in a cross-section of commercial latex medical gloves by using monoclonal and polyclonal antibody-based 2-site immunoenzymetric assays. The overall allergenic potential of these gloves was assessed by IgE-inhibition assay. Stepwise multiple regression analyses were performed to identify marker allergens that best explained the variation in latex glove allergenicity.

RESULTS:
All 6 latex allergens were detected in at least some of the glove samples. Hev b 5 and Hev b 13 were identified as the marker allergens that combined best to explain the variation in the glove allergenicity. The significant multiple correlation (R=0.855) between these 2 markers and glove allergenic potency forms the basis of an assay to gauge latex glove allergenicity.

CONCLUSION:
The overall allergenic potential of latex gloves can be estimated by using Hev b 5 and Hev b 13 as indicator allergens. The correlation between glove allergenicity and the level of these allergens was maintained for low-protein gloves (<200 microg/g). This estimation of glove allergenicity was superior to that obtained by using total protein readings.
The 14.6 kd rubber elongation factor (Hev b 1) and 24 kd (Hev b 3) rubber particle proteins are recognized by IgE from patients with spina bifida and latex allergy

Yeang HY, Cheong KF, Sunderasan E, Hamzah S, Chew NP, Hamid S, et al.

J Allergy Clin Immunol, 1996 Sep;98(3):628-39.
PMID: 8828541 DOI: 10.1016/s0091-6749(96)70097-0

Two major water-insoluble proteins are located on the surface of rubber particles in Hevea brasiliensis latex. A 14.6 kd protein (Hev b 1), found mainly on large rubber particles (> 350 mm in diameter), and a 24 kd protein (Hev b 3), found mainly on small rubber particles (average diameter, 70 nm), are recognized by IgE from patients with spina bifida and latex allergy. Although Hev b 1 (also called the rubber elongation factor [REF]) has previously been reported as a major latex allergen, this conclusion has been disputed on the basis of results from other studies. The allergenicity of Hev b 1 is verified in this study by testing the recombinant protein generated from its gene. Because allergenicity is confined to patients with spina bifida and not observed in adults sensitive to latex, it is not a major latex allergen. The identification of Hev b 3 as another allergen originating from rubber particles is confirmed by immunogold labeling and electron microscopy. Observations with the monoclonal antibody USM/RC2 developed against Hev b 3 show that the protein has a tendency to fragment into several polypeptides of lower molecular weight (from 24 kd to about 5 kd) when stored at -20 degrees C. There is also indication of protein aggregation from the appearance of proteins with molecular weights greater than 24 kd. Fragmentation of Hev b 3 is induced immediately on he addition of latex B-serum, which is normally compartmentalized in the lutoids in fresh latex. In the preparation of ammoniated latex (used for the manufacture of latex products), the lutoids are ruptured, and the released B-serum reacts with Hev b 3 on the rubber particles to give rise to an array of low molecular weight polypeptides that are allergenic to patients with spina bifida.
The manufacture of gloves from natural rubber latex

Yip E, Cacioli P

J Allergy Clin Immunol, 2002 Aug;110(2 Suppl):S3-14.
PMID: 12170237 DOI: 10.1067/mai.2002.124499

Gloves that will provide a barrier of protection from infectious organisms are an essential feature of medical practice for the protection of both patients and medical personnel. Natural rubber latex has consistently been the most satisfactory raw material for the manufacture of gloves. Certain latex proteins, carried over into the finished product by inadequate manufacturing processes, may pose a risk of provoking allergic reactions in some patients and medical workers. As with any allergy, the risk depends on the route of exposure and dose. Hence, the method of manufacture, including the means used to coat gloves to make donning easy, can influence the eventual exposure of sensitive people to latex allergens. In this article, we describe the several processes in use and their effects on latex protein content.