Vaccines against enteric infections for the developing world

Since the first licensure of the Sabin oral polio vaccine more than 50 years ago, only eight enteric vaccines have been licensed for four disease indications, and all are given orally. While mucosal vaccines offer programmatically attractive tools for facilitating vaccine deployment, their development remains hampered by several factors:

• — limited knowledge regarding the properties of the gut immune system during early life;
• — lack of mucosal adjuvants, limiting mucosal vaccine development to live-attenuated or killed whole virus and bacterial vaccines;
• — lack of correlates/surrogates of mucosal immune protection; and
• — limited knowledge of the factors contributing to oral vaccine underperformance in children from developing countries.

There are now reasons to believe that the development of safe and effective mucosal adjuvants and of programmatically sound intervention strategies could enhance the efficacy of current and next-generation enteric vaccines, especially in lesser developed countries which are often co-endemic for enteric infections and malnutrition. These vaccines must be safe and affordable for the world''s poorest, confer long-term protection and herd immunity, and must be able to contain epidemics.     

Center Variation in Intestinal Microbiota Prior to Late-Onset Sepsis in Preterm Infants

Objective

Late onset sepsis (LOS) contributes to mortality and morbidity in preterm infants. We tested the hypotheses that microbes causing LOS originate from the gut, and that distortions in the gut microbial community increases subsequent risk of LOS.

Study Design

We examined the gut microbial community in prospectively collected stool samples from preterm infants with LOS and an equal number of age-matched controls at two sites (Cincinnati, OH and Birmingham, AL), by sequencing the bacterial 16S rDNA. We confirmed our findings in a subset of infants by whole genome shotgun sequencing, and analyzed the data using R and LEfSe.

Results

Infants with LOS in Cincinnati, as compared to controls, had less abundant Actinobacteria in the first samples after birth (median 18 days before sepsis onset), and less abundant Pseudomonadales in the last samples collected prior to LOS (median 8 days before sepsis onset). Infants with LOS in Birmingham, as compared to controls, had no differences identified in the first sample microbial communities, but Lactobacillales was less abundant in the last samples prior to LOS (median 4 days before sepsis onset). Sequencing identified detectable levels of the sepsis-causative organism in stool samples prior to disease onset for 82% of LOS cases.

Conclusions

Translocation of gut microbes may account for the majority of LOS cases. Distortions in the fecal microbiota occur prior to LOS, but the form of distortion depends on timing and site. The microbial composition of fecal samples does not predict LOS onset in a generalizable fashion.     

Short-Term Effect of Antibiotics on Human Gut Microbiota

From birth onwards, the human gut microbiota rapidly increases in diversity and reaches an adult-like stage at three years of age. After this age, the composition may fluctuate in response to external factors such as antibiotics. Previous studies have shown that resilience is not complete months after cessation of the antibiotic intake. However, little is known about the short-term effects of antibiotic intake on the gut microbial community. Here we examined the load and composition of the fecal microbiota immediately after treatment in 21 patients, who received broad-spectrum antibiotics such as fluoroquinolones and β-lactams. A fecal sample was collected from all participants before treatment and one week after for microbial load and community composition analyses by quantitative PCR and pyrosequencing of the 16S rRNA gene, respectively. Fluoroquinolones and β-lactams significantly decreased microbial diversity by 25% and reduced the core phylogenetic microbiota from 29 to 12 taxa. However, at the phylum level, these antibiotics increased the Bacteroidetes/Firmicutes ratio (p = 0.0007, FDR = 0.002). At the species level, our findings unexpectedly revealed that both antibiotic types increased the proportion of several unknown taxa belonging to the Bacteroides genus, a Gram-negative group of bacteria (p = 0.0003, FDR<0.016). Furthermore, the average microbial load was affected by the treatment. Indeed, the β-lactams increased it significantly by two-fold (p = 0.04). The maintenance of or possible increase detected in microbial load and the selection of Gram-negative over Gram-positive bacteria breaks the idea generally held about the effect of broad-spectrum antibiotics on gut microbiota.     

Expression of the Antifeeding Gene anfA1 in Serratia entomophila Requires RpoS

The rpoS gene of Serratia entomophila BC4B was cloned and used to create rpoS-mutant strain BC4BRS. Larvae of the New Zealand grass grub Costelytra zealandica infected with BC4BRS became amber colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene anfA1 in trans was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional rpoS gene is vital for full expression of anfA1 and for development of the antifeeding component of amber disease.     

Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus

In this study, fruit fly of the genus Drosophila is utilized as a suitable model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. Here, we show that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. We also define the few roles of ROS in response to the infection and show that the IMD pathway is required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis.     

Colostrum from Cows Immunized with a Vaccine Associated with Bovine Neonatal Pancytopenia Contains Allo-Antibodies that Cross-React with Human MHC-I Molecules

In 2006, a new haemorrhagic syndrome affecting newborn calves, Bovine Neonatal Pancytopenia (BNP), was reported in southern Germany. It is characterized by severe bleeding, destruction of the red bone marrow, and a high case fatality rate. The syndrome is caused by alloreactive, maternal antibodies that are ingested by the calf with colostrum and result from a dam vaccination with one particular vaccine against Bovine-Viral-Diarrhoea-Virus. Because bovine colostrum is increasingly gaining interest as a dietary supplement for human consumption, the current study was initiated to elucidate whether BNP alloantibodies from BNP dams (i.e. animals that gave birth to a BNP-affected calf) cross-react with human cells, which could pose a health hazard for human consumers of colostral products. The present study clearly demonstrates that BNP alloantibodies cross-react with human lymphocytes in vitro. In agreement with previous reports on BNP, the cross-reactive antibodies are specific for MHC-I molecules, and sensitize opsonised human cells for in vitro complement lysis. Cross-reactive antibodies are present in serum and colostrum of individual BNP dams. They can be traced in commercial colostrum powder manufactured from cows immunized with the vaccine associated with BNP, but are absent from commercial powder manufactured from colostrum excluding such vaccinated cows. In humans alloreactive, MHC-I specific antibodies are generally not believed to cause severe symptoms. However, to minimize any theoretical risk for human consumers, manufacturers of bovine colostrum for human consumption should consider using only colostrum from animals that have not been exposed to the vaccine associated with BNP.     

TAM mediates adaptation of carbapenem-resistant Klebsiella pneumoniae to antimicrobial stress during host colonization and infection

Gram-negative pathogens, such as Klebsiella pneumoniae, remodel their outer membrane (OM) in response to stress to maintain its integrity as an effective barrier and thus to promote their survival in the host. The emergence of carbapenem-resistant K. pneumoniae (CR-Kp) strains that are resistant to virtually all antibiotics is an increasing clinical problem and OM impermeability has limited development of antimicrobial agents because higher molecular weight antibiotics cannot access sites of activity. Here, we demonstrate that TAM (translocation and assembly module) deletion increases CR-Kp OM permeability under stress conditions and enhances sensitivity to high-molecular weight antimicrobials. SILAC-based proteomic analyses revealed mis-localization of membrane proteins in the TAM deficient strain. Stress-induced sensitization enhances clearance of TAM-deficient CR-Kp from the gut lumen following fecal microbiota transplantation and from infection sites following pulmonary or systemic infection. Our study suggests that TAM, as a regulator of OM permeability, represents a potential target for development of agents that enhance the effectiveness of existing antibiotics.     

The tissue localization of Aeromonas salmonicida in rainbow trout, Salmo gairdneri Richardson, following three methods of administration

The localization of a live, and a formalized vaccine preparation of Aeromonas salmonicida within the tissues of rainbow trout, Salmo gairdneri , was followed over a 5 day period. When presented by intraperitoneal injection, both the live bacteria and the vaccine localized in the spleen, liver, kidney and gut. When presented orally, the bacteria and the vaccine were confined almost exclusively to the gut region. Direct immersion resulted in low detectable levels within the tissues, with the spleen and kidney localizing the Aeromonas after its initial attachment to the outer surfaces of the fish.     

NLRP6-associated host microbiota composition impacts in the intestinal barrier to systemic dissemination of Brucella abortus

Brucella abortus is a Gram-negative bacterium responsible for a worldwide zoonotic infection—Brucellosis, which has been associated with high morbidity rate in humans and severe economic losses in infected livestock. The natural route of infection is through oral and nasal mucosa but the invasion process through host gut mucosa is yet to be understood. Studies have examined the role of NLRP6 (NOD-like receptor family pyrin domain-containing-6 protein) in gut homeostasis and defense against pathogens. Here, we investigated the impact of gut microbiota and NLRP6 in a murine model of Ba oral infection. Nlrp6^-/- and wild-type (WT) mice were infected by oral gavage with Ba and tissues samples were collected at different time points. Our results suggest that Ba oral infection leads to significant alterations in gut microbiota. Moreover, Nlrp6^-/- mice were more resistant to infection, with decreased CFU in the liver and reduction in gut permeability when compared to the control group. Fecal microbiota transplantation from WT and Nlrp6^-/- into germ-free mice reflected the gut permeability phenotype from the donors. Additionally, depletion of gut microbiota by broad-spectrum-antibiotic treatment prevented Ba replication in WT while favoring bacterial growth in Nlrp6^-/-. Finally, we observed higher eosinophils in the gut and leukocytes in the blood of infected Nlrp6^-/- compared to WT-infected mice, which might be associated to the Nlrp6^-/- resistance phenotype. Altogether, these results indicated that gut microbiota composition is the major factor involved in the initial stages of pathogen host replication and partially also by the resistance phenotype observed in Nlrp6 -/- mice regulating host inflammation against Ba infection.     

Enteral feeding induces diet-dependent mucosal dysfunction, bacterial proliferation, and necrotizing enterocolitis in preterm pigs on parenteral nutrition

Preterm neonates have an immature gut and metabolism and may benefit from total parenteral nutrition (TPN) before enteral food is introduced. Conversely, delayed enteral feeding may inhibit gut maturation and sensitize to necrotizing enterocolitis (NEC). Intestinal mass and NEC lesions were first recorded in preterm pigs fed enterally (porcine colostrum, bovine colostrum, or formula for 20-40 h), with or without a preceding 2- to 3-day TPN period (n = 435). Mucosal mass increased during TPN and further after enteral feeding to reach an intestinal mass similar to that in enterally fed pigs without TPN (+60-80% relative to birth). NEC developed only after enteral feeding but more often after a preceding TPN period for both sow's colostrum (26 vs. 5%) and formula (62 vs. 39%, both P < 0.001, n = 43-170). Further studies in 3-day-old TPN pigs fed enterally showed that formula feeding decreased villus height and nutrient digestive capacity and increased luminal lactic acid and NEC lesions, compared with colostrum (bovine or porcine, P < 0.05). Mucosal microbial diversity increased with enteral feeding, and Clostridium perfringens density was related to NEC severity. Formula feeding decreased plasma arginine, citrulline, ornithine, and tissue antioxidants, whereas tissue nitric oxide synthetase and gut permeability increased, relative to colostrum (all P < 0.05). In conclusion, enteral feeding is associated with gut dysfunction, microbial imbalance, and NEC in preterm pigs, especially in pigs fed formula after TPN. Conversely, colostrum milk diets improve gut maturation and NEC resistance in preterm pigs subjected to a few days of TPN after birth.     

Antibiotics in Early Life Alter the Gut Microbiome and Increase Disease Incidence in a Spontaneous Mouse Model of Autoimmune Insulin-Dependent Diabetes

Insulin-dependent or type 1 diabetes is a prototypic autoimmune disease whose incidence steadily increased over the past decades in industrialized countries. Recent evidence suggests the importance of the gut microbiota to explain this trend. Here, non-obese diabetic (NOD) mice that spontaneously develop autoimmune type 1 diabetes were treated with different antibiotics to explore the influence of a targeted intestinal dysbiosis in the progression of the disease. A mixture of wide spectrum antibiotics (i.e. streptomycin, colistin and ampicillin) or vancomycin alone were administered orally from the moment of conception, treating breeding pairs, and during the postnatal and adult life until the end of follow-up at 40 weeks. Diabetes incidence significantly and similarly increased in male mice following treatment with these two antibiotic regimens. In NOD females a slight yet not significant trend towards an increase in disease incidence was observed. Changes in gut microbiota composition were assessed by sequencing the V3 region of bacterial 16S rRNA genes. Administration of the antibiotic mixture resulted in near complete ablation of the gut microbiota. Vancomycin treatment led to increased Escherichia, Lactobacillus and Sutterella genera and decreased members of the Clostridiales order and Lachnospiraceae, Prevotellaceae and Rikenellaceae families, as compared to control mice. Massive elimination of IL-17-producing cells, both CD4+TCRαβ+ and TCRγδ+ T cells was observed in the lamina propria of the ileum and the colon of vancomycin-treated mice. These results show that a directed even partial ablation of the gut microbiota, as induced by vancomycin, significantly increases type 1 diabetes incidence in male NOD mice thus prompting for caution in the use of antibiotics in pregnant women and newborns.     

Production and preparation of hyperimmune bovine colostrum for passive immunotherapy of cryptosporidiosis.

Pregnant cows were immunized to produce hyperimmune bovine colostrum (HBC) by intramuscular injection or intramammary infusion (TI) followed by 3 successive TI boosters with Cryptosporidium parvum (Cp) oocyst antigen mixed with Freund's (F) or Ribi (R) adjuvant. Control cows received no Cp. Colostrum from all cows was skimmed of butterfat and tested for specific anti-Cp immunoglobulin isotypes by ELISA. The HBC from Cp-F and Cp-R immunized cows had IgG1 titers exceeding 1:400,000 and 1:800,000, respectively. Some HBC from Cp-F immunized cows was freeze-dried to facilitate storage and some were irradiated at 42.5 kGy to kill potentially contaminating pathogens. Freeze-drying, but not irradiation, reduced IgG1 titers by only one dilution. Neither treatment affected Western blot banding patterns.     

Oral Delivery of a Novel Attenuated Salmonella Vaccine Expressing Influenza A Virus Proteins Protects Mice against H5N1 and H1N1 Viral Infection

Attenuated strains of invasive enteric bacteria, such as Salmonella, represent promising gene delivery agents for nucleic acid-based vaccines as they can be administrated orally. In this study, we constructed a novel attenuated strain of Salmonella for the delivery and expression of the hemagglutinin (HA) and neuraminidase (NA) of a highly pathogenic H5N1 influenza virus. We showed that the constructed Salmonella strain exhibited efficient gene transfer activity for HA and NA expression and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we evaluated the immune responses and protection induced by the constructed Salmonella-based vaccine. Our study showed that the Salmonella-based vaccine induced significant production of anti-HA serum IgG and mucosal IgA, and of anti-HA interferon-γ producing T cells in orally vaccinated mice. Furthermore, mice orally vaccinated with the Salmonella vaccine expressing viral HA and NA proteins were completely protected from lethal challenge of highly pathogenic H5N1 as well as H1N1 influenza viruses while none of the animals treated with the Salmonella vaccine carrying the empty expression vector with no viral antigen expression was protected. These results suggest that the Salmonella-based vaccine elicits strong antigen-specific humoral and cellular immune responses and provides effective immune protection against multiple strains of influenza viruses. Furthermore, our study demonstrates the feasibility of developing novel attenuated Salmonella strains as new oral vaccine vectors against influenza viruses.     

Effect of dietary Bacillus coagulans on the performance and intestinal microbiota of weaned piglets

The performance of weaned piglets suffers from severe limitations resulting from diarrhoea. Therefore, this trial was performed to investigate the effects of Bacillus coagulans as an alternative to antibiotics on piglet growth performance and intestinal health. Ninety (initial BW = 7.70 ± 0.17 kg, weaning age of 26 days) healthy weaned piglets with similar BWs were selected and randomised into three treatment groups. Pigs in the negative control (NC) group were fed a basal diet, pigs in the positive control (PC) group were fed the basal diet plus antibiotics, and pigs in the test group (BC) were fed the basal diet plus Bacillus coagulans at 600 g/t; the trial lasted for 28 days. The results showed that the ratios of feed to gain (F:G) of both the BC and PC groups from 1 to 21 days were significantly lower (P < 0.05), and the average daily weight gain (ADG) of the BC group from 22 to 28 days was significantly higher (P < 0.05) than that of the NC group in terms of growth performance. The diarrhoea index was lowest in the PC group, followed by the BC group, and highest in the NC group. The BC group had a lower diarrhoea index at the later stage. We performed 16S rRNA sequencing to measure the intestinal bacteria and found that the BC group had a higher intestinal bacteria diversity than the NC and PC groups (P < 0.05). From days 1 to 21, the main differential species were Ruminococcaceae_UCG-014 and Faecalibacterium (P < 0.05); from days 22 to 28, the main differential species were Prevotella_9, unclassified_f__Lachnospiraceae, Anaerovibrio, and Ruminococcaceae_UCG-002 (P < 0.05). The correlation analysis between growth performance and species revealed specific gut microorganisms responsible for variation in F:G, ADG, and diarrhoea index, such as Prevotellaceae, Clostridium_sensu_stricto_1, Ruminococcaceae, Phascolarctobacterium, and Anaerovibrio. In conclusion, Bacillus coagulans changed the microbial composition in the faeces of weaned piglets, which had positive effects on growth performance and the diarrhoea index. Therefore, our study provided new insight into the future application of Bacillus coagulans as an additive.     

Intranasal immunization of the combined lipooligosaccharide conjugates protects mice from the challenges with three serotypes of Moraxella catarrhalis

Background

There are no licensed vaccines available against Moraxella catarrhalis, a significant human respiratory pathogen. Lipooligosaccharide (LOS) based conjugate vaccines derived from individual serotype M. catarrhalis only showed partial protection coverage. A vaccine combining LOS conjugates of two or three serotypes might provide a broader protection.

Methods

Mice were immunized intranasally with the combined conjugates consisting of LOS from serotype A and B or serotype A, B, and C followed by challenge with different M. catarrhalis strains of three serotypes. Mouse lungs, nasal washes, and sera were collected after each challenge for bacterial counts, histological evaluation, cytokine profiles, antibody level and binding activity determinations.

Results

Intranasal administration of the combined LOS conjugates not only enhanced pulmonary bacterial clearance of all three serotypes of M. catarrhalis strains in vaccinated mice, but also elevated serotype-specific anti-LOS immunoglobulin (Ig)A and IgG titers in nasal wash and serum respectively. Mice vaccinated with the combined LOS conjugates also showed increased interferon (IFN)-γ, interleukin (IL)-12, and IL-4 in the lungs after challenges. Compared to the control group, mice immunized with the combined LOS conjugates also showed reduced lung inflammation after M. catarrhalis infections. The hyperimmune sera induced by the combined conjugates exhibited a broad cross-reactivity toward all three serotypes of M. catarrhalis under transmission electron microscopy.

Conclusions

The combined vaccine of serotype A and B LOS conjugates provides protection against most M. catarrhalis strains by eliciting humoral and cellular immune responses.     

Intranasal administration of a detoxified endotoxin vaccine protects mice against heterologous Gram-negative bacterial pneumonia

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli J5 mutant lipopolysaccharide (J5dLPS)-group B meningococcal outer membrane protein (-OMP) vaccine previously protected animals from lethal sepsis. To assess the use of this vaccine for the treatment of Gram-negative bacillary pneumonia, we vaccinated mice, with or without the adjuvant CpG, by intranasal (i.n.) or intraperitoneal (i.p.) routes of administration. Local and systemic IgG levels were 2-3 logs higher following i.p. immunization compared to i.n. However, i.n. immunization elicited both local and systemic IgA, unlike i.p. administration. The addition of CpG to the vaccine, by either route of administration, elicited greater levels of antibody. Intranasal immunization protected mice against lethal heterologous Gram-negative bacillary pneumonia and post-immunization serum and broncho-alveolar lavage fluid mediated enhanced bacterial killing with peritoneal and alveolar macrophages in vitro. We conclude that further studies on the use of J5dLPS-OMP for the prevention of nosocomial pneumonia are warranted.     

Questionnaire identifying management practices surrounding calving on spring-calving dairy farms and their associations with herd size and herd expansion

Healthy calves are fundamental to any profitable dairy enterprise. Research to-date, has focused on year-round calving systems which experience many different challenges compared to spring-calving systems. The objective of the present study was to determine the on-farm dry cow, calving, and colostrum management practices of spring-calving dairy production systems, and quantify their associations with herd size and herd expansion status (i.e. expanding or not expanding). Information on these management practices was available from a survey of 262 Irish spring-calving dairy farmers, representative of the Irish national population. Herd expansion in the 2 years before, and the year that the survey was conducted was not associated with any of the management practices investigated. Fifty-three percent of respondents had an average calving season length of 10 to14 weeks with 35% of herds having a longer calving season. Previous research in cattle has documented that both colostrum source and feeding management are associated with the transmission of infectious disease from cow to calf. In the present study 60% of respondents fed calves colostrum from their own dam; however, 66% of those respondents allowed the calf to suckle the dam, 23% of survey respondents fed calves pooled colostrum. Larger herds were more likely (P<0.01) to use pooled colostrum supplies, while smaller herds were more likely (P<0.05) to allow the calf to suckle the dam. The majority (86%) of respondents had stored supplies of colostrum; average-sized herds had the greatest likelihood of storing colostrum (P<0.05), compared to other herd sizes; larger sized herds had a lesser likelihood (P<0.05) of storing colostrum in a freezer, compared to other herd sizes. Although freezing colostrum was the most common method used to store colostrum (54% of respondents), 17% of respondents stored colostrum at room temperature, 29% of which stored it at room temperature for greater than 4 days. The results from the present study indicate that a particular focus needs to be placed on calving and colostrum management because this study has highlighted a number of areas which are below international standards, and may have repercussions for calf health. Furthermore, management practices on larger farms could be improved and, as these represent the future of dairy farming, a focus needs to be placed on them. Expanding herds are not a particular concern as herd expansion, independent of herd size, does not seem to be associated with calving and colostrum management practices on Irish spring-calving dairy herds.     

Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines

Infectious diseases disproportionately affect indigent regions and are the greatest cause of childhood mortality in developing countries. Practical, low-cost vaccines for use in these countries are paramount to reducing disease burdens and concomitant poverty. Algae are a promising low-cost system for producing vaccines that can be orally delivered, thereby avoiding expensive purification and injectable delivery. We engineered the chloroplast of the eukaryotic alga Chlamydomonas reinhardtii to produce a chimeric protein consisting of the 25-kDa Plasmodium falciparum surface protein (Pfs25) fused to the β subunit of the cholera toxin (CtxB) to investigate an alga-based whole-cell oral vaccine. Pfs25 is a promising malaria transmission-blocking vaccine candidate that has been difficult to produce in traditional recombinant systems due to its structurally complex tandem repeats of epidermal growth factor-like domains. The noncatalytic CtxB domain of the cholera holotoxin assembles into a pentameric structure and acts as a mucosal adjuvant by binding GM1 ganglioside receptors on gut epithelial cells. We demonstrate that CtxB-Pfs25 accumulates as a soluble, properly folded and functional protein within algal chloroplasts, and it is stable in freeze-dried alga cells at ambient temperatures. In mice, oral vaccination using freeze-dried algae that produce CtxB-Pfs25 elicited CtxB-specific serum IgG antibodies and both CtxB- and Pfs25-specific secretory IgA antibodies. These data suggest that algae are a promising system for production and oral delivery of vaccine antigens, but as an orally delivered adjuvant, CtxB is best suited for eliciting secretory IgA antibodies for vaccine antigens against pathogens that invade mucosal surfaces using this strategy.     

A common pathway for metabolism of 4-hydroxybenzylglucosinolate in Pieris and Anthocaris (Lepidoptera: Pieridae)

Caterpillars of Pieris rapae L. (Lepidoptera: Pieridae) convert 4-hydroxybenzylglucosinolate (sinalbin) in brassicaceous plants into 4-hydroxybenzylcyanide sulfate (HBC sulfate), with 4-hydroxybenzylcyanide (HBC) as intermediate. This apparently serves as a detoxification, because alternative formation of a mustard oil is avoided. We confirmed the capacity of P. rapae to convert the intermediate HBC into HBC sulfate. Four additional Pieridae – Anthocaris cardamines L., Pieris virginiensis Harris, Pieris napi oleracea Edwards and Pieris brassicae L., likewise excreted HBC sulfate after ingesting leaves with topically added HBC or leaves naturally containing sinalbin and myrosinase, but not after ingesting control leaves devoid of HBC and sinalbin. We confirmed the capacity of the most distantly related pierid species (A. cardamines) for converting ingested (topically added) sinalbin into HBC sulfate. Larvae of two non-pierid Brassicaceae-feeding insects, the oligophagous sawfly Athalia rosae L. (Hymenoptera: Tenthrenidae) and the polyphagous moth Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), did not excrete HBC sulfate after ingesting sinalbin-containing leaves or topically added HBC.