Growth, allergen profile and microbiome studies in Dermatophagoides pteronyssinus cultures

Abstract

Mites are mass-cultured to manufacture allergen extracts for allergy diagnostics and therapeutic treatment. This study focused on characterizing the growth, the allergen profile, and the microbiome of Dermatophagoides pteronyssinus cultures. Mite population, protein profile, total protein content and major allergen levels (Der p 1, Der p 2, Der p 23) were monitored at different times of three independent cultures. The allergenicity was studied by immunoblot using a pool of sera from allergic patients. Mite microbiome was characterized by sequencing the 16S rRNA gene from 600 adult mites from the last day of the culture. Endotoxin content was also analyzed. The cultures had a fast and unrelenting evolution. Mite density, total protein content, major allergen levels and the allergenicity were increased progressively during the cultures. Regarding the microbiome studies, the results confirm the presence of non-pathogenic bacteria, being firmicutes and actinobacteria the most common bacterial taxa, with a very low content of Gram-negative bacteria and endotoxin content. The allergenicity and levels of the main allergens in the mite cultures are objective methods useful to monitor the mite culture that help to produce standardized allergen extracts. The high presence of Gram-positive bacteria found limits the possibility for vaccine contamination by bacterial endotoxins.

Figure 1

Changes in (a) population density (nº mites/mg culture), (b) protein content (ng protein/mg culture) and (c) major allergen levels (ng allergen/mg culture) during culture growth of Dermatophagoides pteronyssinus.

Figure 2

Correlations among the different variables measured in the cultures. (a) Correlation between the mite population (nº mites/mg culture) and total protein content (ng protein/mg culture). (b) Correlations between mite population and the quantities of each of the three major allergens (ng allergen/mg culture). (c) Correlations between the total protein content and the quantities of each of the three major allergens. (d) Correlations between the levels of each pair of major allergens. Pearson r and p-values are shown in each graph.

Figure 3

(a) Protein and (b) allergen profile during the D. pteronyssinus culture. The maximum volume (40 μl) allowed by the system was loaded in each lane. Gels and western blots are original images. Time exposure of blot was 1.5 s.

Figure 4

(a) Phylogenetic assignment of bacterial 16S rRNA gene sequences cloned from mite whole bodies homogenates in D. pteronyssinus samples. (b) Comparative statistical analysis among relative frequency of genus in the three mite cultures.