Cost-effectiveness of patch testing allergens within the same group - a computational approach to optimize formaldehyde-related allergen selection.

BACKGROUND: Patch testing to multiple cross reactive allergens for allergic contact dermatitis (ACD) may not be necessary due to copositivity. OBJECTIVE: We evaluated the formaldehyde group allergens to determine the optimal, most cost-effective allergens to test. METHODS: A retrospective analysis of Mayo Clinic (1997-2022) examined the well-established copositive formaldehyde group: Formaldehyde, Quaternium 15, Hexahydro-1,3,5-tris(2-hydroxyethyl)triazine, Diazolidinyl urea, Imidazolidinyl urea, Toluenesulphonamide formaldehyde resin, DMDM hydantoin, and Ethyleneurea melamine formaldehyde mix. Patch Optimization Platform (POP) identified which single formaldehyde-related allergen optimally captures patients with clinically relevant ACD. Next, POP determined the optimal additional 1, 2, 3, etc. allergens. Cost per patch test was $5.19 (Medicare 2022). RESULTS: 9832 patients were tested to all listed allergens, with 830 having positive patch tests. POP determined that Quaternium 15 alone captures 53% of patients with ACD to the formaldehyde group; adding the optimal second allergen (Formaldehyde 1%) captures 78%; the optimal five top allergens capture over 94% of patients. The incremental cost-per-additional-diagnosis increased up to 44-fold as the number of allergens tested increased. LIMITATIONS: Data is from a single institution, and the cost-per-test was fixed to Medicare Part B in 2022. CONCLUSIONS: For diagnosing ACD, we recommend considering an optimized allergen selection algorithm.

Dengue virus (DV) non-cross-reactive Omicron wave COVID-19 serums enhanced DV3 infectivity in vitro.

Introduction. In India, the SARS-CoV-2 Delta wave (2020-2021) faded away with the advent of the Omicron variants (2021-present). Dengue incidences were observed to be less in Southeast Asia during the active years of the pandemic (2020-2021). However, dengue virus type 3 (DV3) cases were increasingly reported in this region (including India) concurrent with the progression of the Omicron waves since 2022.Hypothesis. What could be the reason(s) behind this unusual DV3 surge after an overall dip in dengue incidences in many parts of Southeast Asia?Aim. We, therefore, investigated the current state of cross-reactivity of prevalent (Omicron era) SARS-CoV-2 serums with different DV serotypes and evaluated the impact of such serums on DV neutralization in cell culture.Methodology. Fifty-five COVID-19 serum samples (January-September 2022) and three pre-pandemic archived serum samples from apparently healthy individuals were tested for DV or SARS-CoV-2 IgM/IgG using the lateral flow immunoassays. DV1-4 virus neutralization tests (VNTs) were done with the SARS-CoV-2 antibody (Ab)-positive serums in Huh7 cells. DV3 envelope (env) gene was PCR amplified and sequenced for three archived DV isolates, one from 2017 and two from 2021.Results. SARS-CoV-2 Ab-positive samples constituted 74.5 % of the serums. Of these, 41.5 % were DV cross-reactive and 58.5 % were not. The DV cross-reactive serums neutralized all DV serotypes (DV1-4), as per previous results and this study. The DV non-cross-reactive serums (58.5 %) also cross-neutralized DV1, 2 and 4 but increased DV3 infectivity by means of antibody-dependent enhancement of infection as evident from significantly higher DV3 titres in VNT compared to control serums. The DV3 envelope was identical among the three isolates, including isolate 1 used in VNTs. Our results suggest that DV cross-reactivity of SARS-CoV-2 serums diminished with the shift from Delta to Omicron prevalence. Such COVID-19 serums (DV non-cross-reactive) might have played a major role in causing DV3 surge during the Omicron waves.Conclusion. Patients suspected of dengue or COVID-19 should be subjected to virus/antigen tests and serological tests for both the diseases for definitive diagnosis, prognosis and disease management.

Therapeutic antibodies: technical points to consider in tissue cross-reactivity studies.

Tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies are conducted to estimate any possible binding sites within the human body that can be affected by the antibody when assessing safety in humans. Any possible binding sites include specific binding sites of the antibody to its target antigen and nonspecific or off-target binding sites. In TCR studies the therapeutic antibodies and immunohistochemistry (IHC) of frozen tissues must be applied in assays. However, there are technical issues with applying a therapeutic antibody or test article to IHC, such as human-on-human staining, difficulty in applying the test article to IHC, and retention of the target antigen in frozen sections. In the current review, we introduce three case studies in which these technical issues were addressed, and propose a practical scheme for points to consider when conducting a TCR study. Information on the target antigen distribution obtained through robust assays and case-by-case strategies were found to be useful for understanding and assessing the relevance of toxic effects between animals and humans. Thus, we anticipate that by considering the points discussed in the current review and combining the data with information on the biological features of the target antigens and therapeutic antibodies, it will be possible to predict safety risks in humans with higher accuracy.

Neglected but Clinically Relevant Allergens in Korea.

PURPOSE OF REVIEW: Allergy diagnostics and immunotherapeutics in Asia heavily rely on imported products from Western countries, raising concerns about the accuracy and efficacy of these products for the management of Asian allergy patients. RECENT FINDINGS: Recent advancements in allergen research have led to the identification and characterization of novel allergens from indigenous Korean species. While some allergens share homology with well-known allergens, others lack counterparts in imported allergen extracts. Classifying regional allergens in Asia into three categories based on their cross-reactivity with imported allergens offers valuable insights. Highly cross-reactive allergens, such as oak allergens Que m 1 from Quercus mongolica and Que ac 1 from Q. acutissima, can be effectively substituted with the imported allergens. Allergens with partial cross-reactivity, like the Asian needle ant allergen Pac c 3 (Antigen 5), permit limited diagnostic value by the currently available products. Unique allergens, including the Japanese hop allergen Hum j 6 (pectin methylesterase inhibitor) and the silkworm pupa allergen Bomb m 4 (30 kDa hemolymph lipoprotein) lack alternatives in the available product list. Greater attention is needed, particularly for species listed as ecologically invasive in Western regions. Additionally, allergens from domestic fruits and vegetables causing pollen food allergy syndrome require characterization for the development of improved diagnostics.

Insights into insulin analog cross-reactivity: a comparative study of Siemens Atellica and LC-MS/MS.

BACKGROUND: To address the challenges posed by inconsistent detection of analog insulin in commercially available insulin immunoassays, resulting in potential discrepancies in clinical findings and misdiagnosis during the investigation of factitious hypoglycemia., we aimed to evaluate the ability of the Siemens Atellica automated immunoassay to detect insulin analogs compared with LC-MS/MS. METHODS: Five insulin analogs were analyzed at 10 ng/mL spiked into serum samples, with recombinant human insulin as positive controls. Insulin and C-peptide assays were performed using Siemens Atellica and LC-MS/MS. Recovery rates were calculated. RESULTS: Siemens Atellica immunoassay demonstrated robust cross-reactivity (92-121%) of insulin analogs. In contrast, glargine was detected by LC-MS/MS but other analogs were not observed (<10% recovery). CONCLUSION: Our results indicate that the insulin assay conducted on the Siemens Atellica platform could be used to diagnose factitious hypoglycemia by detecting the specific insulin analogs involved. The findings from our studies indicate the suitability of this method for clinical laboratory use in cases where factitious hypoglycemia is under consideration as a potential diagnosis. Clinicians should take these results into account when interpreting insulin measurements, particularly in instances where insulin analog overdose is suspected.

Fenugreek allergy caused by cross-reactivity with peanut: An in vitro analysis.

Fenugreek is often hidden in processed foods, where it is included in the generic term spice on food labels. Cross-reactivity of peanut and fenugreek should be considered in the management of peanut allergy.

IgE-Mediated Allergy and Asymptomatic Sensitization to Cannabis Allergens-Review of Current Knowledge and Presentation of Six Cases.

Cannabis allergy is a relatively new phenomenon described in the 1970s. Its increased frequency has been observed over the last years due to the increasing therapeutic and recreational use of cannabis-based products. Sensitization possibly leading to allergy symptoms can occur not only through the smoking of cannabis, but also through ingestion, the inhalation of pollen, or direct contact. The severity of symptoms varies from benign pruritus to anaphylaxis. There is scant information available to support clinicians throughout the entire therapeutic process, starting from diagnosis and ending in treatment. In this review, we present six cases of patients in whom molecular in vitro testing revealed sensitization to cannabis extract and/or cannabis-derived nsLTP molecules (Can s 3). Based on these cases, we raise important questions regarding this topic. The article discusses current proposals and highlights the importance of further research not only on cannabis allergy but also on asymptomatic sensitization to cannabis allergens, which may be ascertained in some percentage of the population.

Cross-Reactivity of Ragweed Pollen Calcium-Binding Proteins and IgE Sensitization in a Ragweed-Allergic Population from Western Romania.

Ragweed pollen allergy is the most common seasonal allergy in western Romania. Prolonged exposure to ragweed pollen may induce sensitization to pan-allergens such as calcium-binding proteins (polcalcins) and progression to more severe symptoms. We aimed to detect IgE sensitization to recombinant Amb a 9 and Amb a 10 in a Romanian population, to assess their potential clinical relevance and cross-reactivity, as well as to investigate the relation with clinical symptoms. rAmb a 9 and rAmb a 10 produced in Escherichia coli were used to detect specific IgE in sera from 87 clinically characterized ragweed-allergic patients in ELISA, for basophil activation experiments and rabbit immunization. Rabbit rAmb a 9- and rAmb a 10-specific sera were used to detect possible cross-reactivity with rArt v 5 and reactivity towards ragweed and mugwort pollen extracts. The results showed an IgE reactivity of 25% to rAmb a 9 and 35% to rAmb a 10. rAmb a 10 induced basophil degranulation in three out of four patients tested. Moreover, polcalcin-negative patients reported significantly more skin symptoms, whereas polcalcin-positive patients tended to report more respiratory symptoms. Furthermore, both rabbit antisera showed low reactivity towards extracts and showed high reactivity to rArt v 5, suggesting strong cross-reactivity. Our study indicated that recombinant ragweed polcalcins might be considered for molecular diagnosis.

Development of an indirect competitive ELISA based on the common epitope of fish parvalbumin for its detection.

A common epitope (AGSFDHKKFFKACGLSGKST) of parvalbumin from 16 fish species was excavated using bioinformatics tools combined with the characterization of fish parvalbumin binding profile of anti-single epitope antibody in this study. A competitive enzyme-linked immunosorbent assay (ELISA) based on the common epitope was established with a limit of detection of 10.15 ng/mL and a limit of quantification of 49.29 ng/mL. The developed ELISA exhibited a narrow range (71% to 107%) of related cross-reactivity of 15 fish parvalbumin. Besides, the recovery, the coefficient of variations for the intra-assay and the inter-assay were 84.3% to 108.2%, 7.4% to 13.9% and 8.5% to 15.6%. Our findings provide a novel idea for the development of a broad detection method for fish allergens and a practical tool for the detection of parvalbumin of economic fish species in food samples.

Remimazolam anaphylaxis in a patient not allergic to brotizolam: a case report and literature review.

BACKGROUND: Remimazolam is a recently developed, ultrashort-acting benzodiazepine that is used as a general anesthetic. Some cases of remimazolam anaphylaxis have been reported, but its characteristics are not fully understood. We present an interesting case report and review of the literature to better understand remimazolam anaphylaxis. CASE PRESENTATION: A 75-year-old man scheduled for robot-assisted gastrectomy was administered remimazolam for the induction of general anesthesia. After intubation, low end-expiratory CO2, high airway pressure and concurrent circulatory collapse were observed. Bronchoscopy revealed marked tracheal and bronchial edema, which we diagnosed as anaphylaxis. The patient suffered cardiac arrest after bronchoscopy but recovered immediately with intravenous adrenaline administration and chest compressions. We performed skin prick tests for the drugs used during induction except for remimazolam, considering the high risk of systemic adverse reactions to remimazolam. We diagnosed remimazolam anaphylaxis because the skin prick test results for the other drugs used during anesthesia were negative, and these drugs could have been used without allergic reactions during the subsequent surgery. Furthermore, this patient had experienced severe anaphylactic-like reactions when he underwent cardiac surgery a year earlier, in which midazolam had been used, but it was not thought to be the allergen at that time. Based on these findings, cross-reactivity to remimazolam and midazolam was suspected. However, the patient had previously received another benzodiazepine, brotizolam, to which he was not allergic, suggesting that cross-reactivity of remimazolam may vary among benzodiazepines. In this article, we reviewed the 11 cases of remimazolam anaphylaxis that have been described in the literature. CONCLUSIONS: Remimazolam is an ultrashort-acting sedative; however, it can cause life-threatening anaphylaxis. In addition, its cross-reactivity with other benzodiazepines is not fully understood. To increase the safety of this drug, further research and more experience in its use are needed.

Comparative functional characterization and in vitro immunological cross-reactivity studies on Daboia russelii and Craspedocephalus malabaricus venom.

BACKGROUND: Snake venom is a complex mixture of organic and inorganic constituents, including proteins and peptides. Several studies showed that antivenom efficacy differs due to intra- and inter-species venom variation. METHODS: In the current study, comparative functional characterization of major enzymatic proteins present in Craspedocephalus malabaricus and Daboia russelii venom was investigated through various in vitro and immunological cross-reactivity assays. RESULTS: The enzymatic assays revealed that hyaluronidase and phospholipase A2 activities were markedly higher in D. russelii. By contrast, fibrinogenolytic, fibrin clotting and L-amino acid oxidase activities were higher in C. malabaricus venom. ELISA results suggested that all the antivenoms had lower binding potential towards C. malabaricus venom. For D. russelii venom, the endpoint titration value was observed at 1:72 900 for all the antivenoms. In the case of C. malabaricus venom, the endpoint titration value was 1:2700, except for Biological E (1:8100). All these results, along with the avidity assays, indicate the strength of venom-antivenom interactions. Similarly, the western blot results suggest that all the antivenoms showed varied efficacies in binding and detecting the venom antigenic epitopes in both species. CONCLUSIONS: The results highlight the need for species-specific antivenom to better manage snakebite victims.

Management and Implications of Beta-Lactam Allergies.

Beta-lactam antibiotics are essential components in the current antimicrobial treatment strategy, playing a crucial role in ambulatory patients and hospitalized patients. Despite their prominent therapeutic index, the use of beta-lactam can lead to adverse effects, with allergic reactions being the most concerning because of their severity. Additionally, the phenomenon of cross-reactivity may occur among various beta-lactam families, with side chains significantly contributing to immunological recognition, making these structures often responsible for the cross-allergic reactivity of beta-lactams. Tools to assess beta-lactam allergy include taking a patient's medical history, performing skin tests, and conducting provocation tests. This research aims to analyze the relevant aspects related to the safe administration of beta-lactam antibiotics in hospitalized patients as well as provide knowledge on the proper management of patients with such hypersensitivity, by doing systemic research. This research was made using Google Scholar and keywords such as "Beta-lactam allergy," "Hypersensitivity," "Cross-reactivity," "Desensitization," and "Beta-lactam allergy management." In conclusion, substituting a beta-lactam antibiotic with an alternative antibiotic may not always be the best management option for these patients, as it may lead to more adverse effects, be less effective, and prolong hospitalization time. It may also result in higher rates of antibiotic-resistant infections and increased medical costs, as these alternatives are often more expensive. However, an alternative within the beta-lactam family can be sought by conducting the appropriate analyses. Although cross-reactivity does not always occur among all beta-lactams, potential cross-reactivity should always be considered.

A highly effective ferritin-based divalent nanoparticle vaccine shields Syrian hamsters against lethal Nipah virus.

The Nipah virus (NiV), a highly deadly bat-borne paramyxovirus, poses a substantial threat due to recurrent outbreaks in specific regions, causing severe respiratory and neurological diseases with high morbidity. Two distinct strains, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), contribute to outbreaks in different geographical areas. Currently, there are no commercially licensed vaccines or drugs available for prevention or treatment. In response to this urgent need for protection against NiV and related henipaviruses infections, we developed a novel homotypic virus-like nanoparticle (VLP) vaccine co-displaying NiV attachment glycoproteins (G) from both strains, utilizing the self-assembling properties of ferritin protein. In comparison to the NiV G subunit vaccine, our nanoparticle vaccine elicited significantly higher levels of neutralizing antibodies and provided complete protection against a lethal challenge with NiV infection in Syrian hamsters. Remarkably, the nanoparticle vaccine stimulated the production of antibodies that exhibited superior cross-reactivity to homologous or heterologous henipavirus. These findings underscore the potential utility of ferritin-based nanoparticle vaccines in providing both broad-spectrum and long-term protection against NiV and emerging zoonotic henipaviruses challenges.

Autoantibodies inhibit Plasmodium falciparum growth and are associated with protection from clinical malaria.

Many infections, including malaria, are associated with an increase in autoantibodies (AAbs). Prior studies have reported an association between genetic markers of susceptibility to autoimmune disease and resistance to malaria, but the underlying mechanisms are unclear. Here, we performed a longitudinal study of children and adults (n = 602) in Mali and found that high levels of plasma AAbs before the malaria season independently predicted a reduced risk of clinical malaria in children during the ensuing malaria season. Baseline AAb seroprevalence increased with age and asymptomatic Plasmodium falciparum infection. We found that AAbs purified from the plasma of protected individuals inhibit the growth of blood-stage parasites and bind P. falciparum proteins that mediate parasite invasion. Protected individuals had higher plasma immunoglobulin G (IgG) reactivity against 33 of the 123 antigens assessed in an autoantigen microarray. This study provides evidence in support of the hypothesis that a propensity toward autoimmunity offers a survival advantage against malaria.

Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals.

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

Evidences support that dengue virus can impart broad-spectrum immunity against betacoronaviruses in dengue endemic regions.

COVID-19 tended to be less aggressive in dengue endemic regions. Conversely, dengue cases plummeted in dengue endemic zones during the active years of the pandemic (2020-2021). We and others have demonstrated serological cross-reactivity between these two viruses of different families. We further demonstrated that COVID-19 serum samples that were cross-reactive in dengue virus (DV) serological tests, "cross-neutralized" all DV serotypes in Huh7 cells. Here we showed by co-immunoprecipitation (Co-IP) and atomic force microscopy (AFM) imaging that severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 (SARS-CoV-2) spike (S) protein subunit S1 and S2 monoclonal antibodies can indeed, bind to DV particles. Likewise, DV envelope antibodies (DV E Abs) showed high docking frequency with other human pathogenic beta-CoVs and murine hepatitis virus-1 (MHV-1). SARS-CoV-2 Ab didn't show docking or Co-IP with MHV-1 supporting poor cross-protection among CoVs. DV E Abs showed binding to MHV-1 (AFM, Co-IP, and immunofluorescence) and prepandemic dengue patients' serum samples even "cross-neutralized" MHV-1 plaques in cell culture. Furthermore, dengue serum samples showed marked inhibition potential in a surrogate virus-based competitive enzyme-linked immunosorbent assay, used for determining neutralizing Abs against SARS-CoV-2 S protein receptor-binding domain in COVID-19 serum samples. We therefore, provide multiple evidence as to why CoVs are epidemiologically less prevalent in highly dengue endemic regions globally.

Anti-RBD IgG antibodies from endemic coronaviruses do not protect against the acquisition of SARS-CoV-2 infection among exposed uninfected individuals.

Background: The Coronaviridae family comprises seven viruses known to infect humans, classified into alphacoronaviruses (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1), which are considered endemic. Additionally, it includes SARS-CoV (severe acute respiratory syndrome), MERS-CoV (Middle East respiratory syndrome), and the novel coronavirus SARS-CoV-2, responsible for COVID-19. SARS-CoV-2 induces severe respiratory complications, particularly in the elderly, immunocompromised individuals and those with underlying diseases. An essential question since the onset of the COVID-19 pandemic has been to determine whether prior exposure to seasonal coronaviruses influences immunity or protection against SARS-CoV-2. Methods: In this study, we investigated a cohort of 47 couples (N=94), where one partner tested positive for SARS-CoV-2 infection via real-time PCR while the other remained negative. Plasma samples, collected at least 30 days post-PCR reaction, were assessed using indirect ELISA and competition assays to measure specific antibodies against the receptor-binding domain (RBD) portion of the Spike (S) protein from SARS-CoV-2, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1. Results: IgG antibody levels against the four endemic coronavirus RBD proteins were similar between the PCR-positive and PCR-negative individuals, suggesting that IgG against endemic coronavirus RBD regions was not associated with protection from infection. Moreover, we found no significant IgG antibody cross-reactivity between endemic coronaviruses and SARS-CoV-2 RBDs. Conclusions: Taken together, results suggest that anti-RBD antibodies induced by a previous infection with endemic HCoVs do not protect against acquisition of COVID-19 among exposed uninfected individuals.

Cracking the antigenic code of mycobacteria: CFP-10/ESAT-6 tuberculosis skin test and misleading results.

There are different tuberculosis diagnostic tools available that detect an antigen-specific immune response. The present study aims to evaluate the potential of cross-reactive responses of a CFP-10 and ESAT-6 antigen-based TB test using bioinformatics tools. The study found that the presence of the sequences coding for the CFP-10 and ESAT-6 antigens in mycobacterial genomes is not associated with their pathogenicity, and not even consistent within a single species among its strains, which can lead to either false positive or false negative test results. The data that was analyzed included genome assemblies of all available mycobacterial strains obtained from the NCBI Genome database, while the standalone BLAST and tblastn programs were utilized to detect the presence of the CFP-10 and ESAT-6 sequences. The findings revealed that a number of non-pathogenic mycobacteria contained the aforementioned sequences, while some pathogenic mycobacteria did not, indicating that a standard tuberculin skin test should be more preferable for detecting various pathogenic mycobacteria compared to antigen-specific tests. In the Mycobacterium tuberculosis complex (MTBC), the proportion of positive strains varied within individual species, indicating a complex relationship. Among non-tuberculous mycobacteria (NTMB), more than half of the analyzed species did not contain these sequences which is consistent with their non-pathogenicity. Further research is necessary to fully comprehend the relationship between MTBC pathogenicity and the CFP-10 and ESAT-6 sequences. This could lead to a conclusion that a standard tuberculin skin test, although non-specific due to the undefined antigen content, may be able to detect various pathogenic mycobacteria in a more reliable manner than antigen-specific tests.

Conservation of C4BP-binding sequence patterns in Streptococcus pyogenes M and Enn proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Impact of processing on the sensitising capacity and cross-reactivity of cow's and camel milk proteins in a Brown Norway rat study.

Infant formulas based on hydrolysed cow's milk proteins are used when breastfeeding is not feasible in cow's milk allergic infants. Camel milk has been shown to be well-tolerated by the majority of children with cow's milk allergy (CMA) and may be a substitute in management of CMA. Here we aimed to evaluate the impact of processing on immunogenicity, sensitising, antibody-binding and cross-reactive capacity of cow's and camel milk. Cow's and camel milk were processed by means of enzyme hydrolysis or heat treatment. Brown Norway rats were immunised with PBS, non-processed, enzyme hydrolysed or heat-treated cow's or camel milk. In vivo tests were performed for evaluation of clinical signs. Blood and faecal samples were analysed for levels and specificity of antibody responses. Cow's and camel milk showed similar sensitising capacity. Processing decreased the sensitising capacity of cow's milk, yet only enzyme hydrolysis but not heat treatment decreased the sensitising capacity of camel milk. Processing affected the specificity of antibodies raised in the rats, though the effect differed between cow's and camel milk. The study showed a low cross-reactivity between cow's and camel milk, which was decreased with processing, suggesting that processing of camel milk may improve its usefulness in CMA management.