In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay (“NeutrobodyPlex”) for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.

Crossbreeding in dairy cattle has been used to improve functional traits, milk composition, and efficiency of Holstein herds. The objective of the study was to compare indicators of the metabolic energy balance, nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), glucose, body condition score (BCS) back fat thickness (BFT), as well as milk yield and milk composition of Holstein and Simmental cows, and their crosses from the prepartum period until the 100th day of lactation at the Livestock Center of the Ludwig Maximilians University (Munich, Germany). In total, 164 cows formed five genetic groups according to their theoretic proportion of Holstein and Simmental genes as follows: Holstein (100% Holstein; n = 9), R1-Hol (51–99% Holstein; n = 30), first generation (F1) crossbreds (50% Holstein, 50% Simmental; n = 17), R1-Sim (1–49% Holstein; n = 81) and Simmental (100% Simmental; n = 27). The study took place between April 2018 and August 2019. BCS, BFT blood parameters, such as BHBA, glucose, and NEFA were recorded weekly. A mixed model analysis with fixed effects breed, week (relative to calving), the interaction of breed and week, parity, calving year, calving season, milking season, and the repeated measure effect of cow was used. BCS increased with the Simmental proportion. All genetic groups lost BCS and BFT after calving. Simmental cows showed lower NEFA values. BHBA and glucose did not differ among genetic groups, but they differed depending on the week relative to calving. Simmental and R1-Sim cows showed a smaller effect than the other genetic groups regarding changes in body weight, BCS, or back fat thickness after a period of a negative energy balance after calving. There was no significant difference for milk yield among genetic groups, although Simmental cows showed a lower milk yield after the third week after calving. Generally, Simmental and R1-Simmental cows seemed to deal better with a negative energy balance after calving than purebred Holstein and the other crossbred lines. Based on a positive heterosis effect of 10.06% for energy corrected milk (ECM), the F1, however, was the most efficient crossbred line.

Research has shown that digestion of A1 β-casein (β-CN) affects gastrointestinal motility and opioid activity through the release of the peptide β-casomorphin-7 (β-CM7). In the case of the A2 variant, the cleavage of β-CM7 does not occur or occurs at a very low rate. Therefore, the aim of the study was to compare the effects of milk containing either homozygote A1 or A2 β-CN on health and growth parameters of dairy calves. Forty-seven neonatal calves (24 females, 23 males) of the breeds German Holstein (GH, n = 9), German Simmental (GS, n = 33) and their crossing (GH × GS, n = 5) were used in a 21-day feeding study. Fecal score (FS), respiratory frequency (RF), and rectal body temperature (BT) were recorded daily, whereas body weight was measured at birth and at day 21 to estimate the average daily weight gain (ADG). Additionally, blood was collected from calves three times during the experimental period and, for the first time, the respective plasma samples were analyzed for intact β-CM7. Consumption of A2-milk led to a lower daily milk intake (dMI) (p < 0.05). Furthermore, fecal consistency was softer for calves fed A2-milk (p < 0.05). Although 44% of A2-calves had diarrhea or revealed a tendency towards it (FS ≥ 3), A1-calves had a prevalence of 21%. Calves with a FS of 4 were offered an electrolyte solution and received a dietary food supplement for the stabilization of the fluid and electrolyte balance. Nevertheless, similar ADG and end weights (EW) of calves fed A1- or A2-milk (p > 0.05) indicate that A2-milk may compensate higher diarrhea rates and lower dMI due to the associated higher protein content. This is the first report of intact β-CM7 in plasma of calves fed milk of either A1 or A2 β-CN. Evidence from this study suggests that due to the change in the amino-acid sequence, A2-milk might be able to prevent or, at least, to minimize the cleavage of β-CM7 in calves.

The objective of this study was to phenotype visceral adipose tissue (VAT) in pigs. In this context, the ability to detect VAT by using the DXA CoreScan mode within the enCORE software, version 17 (GE Healthcare) was evaluated in comparison with MRI measurements (Siemens Magnetom C!) of the same body region. A number of 120 crossbred pigs of the F1 and F2 generation, with the parental breeds Large White, Landrace, Piétrain, and Duroc, were examined at an age of 150 days. A whole-body scan in two different modes (“thick”, “standard”) was carried out by a GE Lunar iDXA scanner. Very strong relationships (R² = 0.95, RMSE = 175 cm³) were found for VAT between the two DXA modes. The comparison of VAT measured by MRI and DXA shows high linear relationships (“thick”: R² = 0.76, RMSE = 399.25 cm³/“standard”: R² = 0.71, RMSE = 443.42 cm³), but is biased, according to the Bland–Altman analysis. A variance analysis of VAT shows significant differences for both DXA modes and for MRI between male and female pigs, as well as between F1 and F2. In conclusion, DXA “CoreScan” has the ability to estimate VAT in pigs with a close relationship to MRI but needs bias correction.

The aim of this study was to compare the effects of feeding homozygous β-CN A1 or A2 milk on the body composition, milk intake, and growth of German Holstein (GH), German Simmental (GS), and crossbred (CR) dairy calves of both sexes during the first 2 wk of life. A total of 104 calves (n = 54 female, f; and n = 50 male, m) from the breed groups GH (n = 23), GS (n = 61), and crossbred GH × GS (n = 20) were evaluated. Calves were weighed after birth and received colostrum ad libitum. On the second day, calves were alternately housed in pairs in double-igloo systems according to their random birth order and received either A1 milk (n = 52; 27 female and 25 male) or A2 milk (n = 52; 27 female and 25 male). They were offered 7.5 L/d, and the individual actual total milk intake was recorded. Daily energy-corrected milk intake was also calculated based on the milk composition (fat and protein). Fecal scores were recorded daily. On d 15, visceral adipose tissue (VAT) volume was assessed by open magnetic resonance imaging and dual-energy X-ray absorptiometry (DXA). In addition, fat and lean mass (g), as well as bone mineral content (g) and bone mineral density (g/cm²), were determined by DXA. The body composition, milk intake, and growth were similar between the 2 types of milk in the first 2 wk of life. Female calves had more VAT and fat mass, but less lean mass than male calves. GH and CR calves had more VAT and less lean mass than GS calves. Male calves were heavier than female calves after birth and on d 15. The average days with diarrhea and diarrhea occurrence were similar between calves fed A1 and A2 milk and between both sex groups. GS calves presented slightly more days with diarrhea and increased odds of having diarrhea compared with GH calves, not differing from CR.

As the COVID-19 pandemic escalates, the need for effective vaccination programs, therapeutic intervention, and diagnostic tools increases. Here, we identified 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD) of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following a detailed epitope determination and characterization of the binding mode by structural analysis, we constructed a hetero-bivalent Nb targeting two different epitopes within the RBD:ACE2 interface. This resulted in a high-affinity binder with a viral neutralization efficacy in the picomolar range. Using the bivalent Nb as a surrogate, we established a competitive multiplex binding assay (“NeutrobodyPlex”) for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in the serum of convalescent or vaccinated patients. As demonstrated, the NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. This approach is easily transferrable to diagnostic laboratories worldwide.