Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewisx antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.

Obesity is the largest risk factor for the development of chronic diseases in industrialized countries. Excessive fat accumulation triggers a state of chronic low-grade inflammation to the detriment of numerous organs. To address this problem, our lab has been examining the anti-inflammatory mechanisms of two human milk oligosaccharides (HMOs), lacto-N-fucopentaose III (LNFPIII) and lacto-N-neotetraose (LNnT). LNFPIII and LNnT are HMOs that differ in structure via presence/absence of an α1,3-linked fucose. We utilize LNFPIII and LNnT in conjugate form, where 10-12 molecules of LNFPIII or LNnT are conjugated to a 40 kDa dextran carrier (P3DEX/NTDEX). Previous studies from our lab have shown that LNFPIII conjugates are anti-inflammatory, act on multiple cell types, and are therapeutic in a wide range of murine inflammatory disease models. The α1,3-linked fucose residue on LNFPIII makes it difficult and more expensive to synthesize. Therefore, we asked if LNnT conjugates induced similar therapeutic effects to LNFPIII. Herein, we compare the therapeutic effects of P3DEX and NTDEX in a model of diet-induced obesity (DIO). Male C57BL/6 mice were placed on a high-fat diet for six weeks and then injected twice per week for eight weeks with 25µg of 40 kDa dextran (DEX; vehicle control), P3DEX, or NTDEX. We found that treatment with P3DEX, but not NTDEX, led to reductions in body weight, adipose tissue (AT) weights, and fasting blood glucose levels. Mice treated with P3DEX also demonstrated improvements in glucose homeostasis and insulin tolerance. Treatment with P3DEX or NTDEX also induced different profiles of serum chemokines, cytokines, adipokines, and incretin hormones, with P3DEX notably reducing circulating levels of leptin and resistin. P3DEX also reduced WAT inflammation and hepatic lipid accumulation, whereas NTDEX seemed to worsen these parameters. These results suggest that the small structural difference between P3DEX and NTDEX has significant effects on the conjugates’ therapeutic abilities. Future work will focus on identifying the receptors for these conjugates and delineating the mechanisms by which P3DEX and NTDEX exert their effects.

Accurate measures of nematode fecundity can provide important information for investigating parasite life history evolution, transmission potential, and effects on host health. Understanding differences among fecundity assessment protocols and standardizing methods, where possible, will enable comparisons across different studies and host and parasite species and systems. Using the trichostrongyle nematode Cooperia fuelleborni isolated from wild African buffalo (Syncerus caffer), we compared egg recovery and enumeration between two methods for measuring the fecundity of female worms. The first method, in utero egg count, involves visual enumeration of the eggs via microscopic inspection of the uterine system. The second method, ex utero egg count, involves dissolving the same specimens from above in a sodium chloride solution to release the eggs from the female's uterus, then enumeration under an inverted microscope. On average, the ex utero method resulted in 34% more eggs than the in utero method. However, results indicate that the two methods used to quantify parasitic nematode fecundity are highly correlated. Thus, while both methods are viable options for estimating relative nematode fecundity, we recommend caution in undertaking comparative studies that utilize egg count data collected using different methods.