A rational engineering strategy for designing protein a-binding camelid single-domain antibodies

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species

Effect of plasma sodium concentration on blood pressure regulators during hemodialysis:A randomized crossover study

Background: Intradialytic hypotension is a common complication of hemodialysis. The Hemocontrol biofeedback system, improving intradialytic hemodynamic stability, is associated with an initial transient increase in plasma sodium levels. Increases in sodium could affect blood pressure regulators. Methods: We investigated whether Hemocontrol dialysis affects vasopressin and copeptin levels, endothelial function, and sympathetic activity in twenty-nine chronic hemodialysis patients. Each patient underwent one standard hemodialysis and one Hemocontrol hemodialysis. Plasma sodium, osmolality, nitrite and nitrate (NOx), endothelin-1, angiopoietins-1 and 2, and methemoglobin as measures of endothelial function, plasma catecholamines as indices of sympathetic activity and plasma vasopressin and copeptin levels were measured six times during each modality. Blood pressure, heart rate, blood volume, and heart rate variability were repeatedly monitored. Generalized Estimating Equations was used to compare the course of the parameters during the two treatment modalities. Results: Plasma sodium and osmolality were significantly higher during the first two hours of Hemocontrol hemodialysis. Overall, mean arterial pressure (MAP) was higher during Hemocontrol dialysis. Neither the measures of endothelial function and sympathetic activity nor copeptin levels differed between the two dialysis modalities. In contrast, plasma vasopressin levels were significantly higher during the first half of Hemocontrol dialysis. The intradialytic course of vasopressin was associated with the course of MAP. Conclusions: A transient intradialytic increase in plasma sodium did not affect indices of endothelial function or sympathetic activity compared with standard hemodialysis, but coincided with higher plasma vasopressin levels. The beneficial effect of higher intradialytic sodium levels on hemodynamic stability might be mediated by vasopressin

Mutation in the Fas Pathway Impairs CD8+ T Cell Memory.

In press: YesNRC publication: Ye

Specificity analysis of the C-type lectin from rattlesnake venom, and its selectivity towards Gal- or GalNAc-terminated glycoproteins

The rattlesnake (Crotalus atrox) venom lectin is a readily-prepared decameric C-type lectin, specific for Gal and GalNAc. Glycan microarray analysis showed it reacted with a wide range of glycans, chiefly recognizing sets of compounds with Gal\u3b21-4GlcNAc (LacNAc), \u3b1-Gal or \u3b1-GalNAc non-reducing termini. Its array profile was therefore distinctly different from those of four previously studied mammalian C-type lectins with the same Gal/GalNAc monosaccharide specificity, and it was more broadly reactive than several Gal- or GalNAc-specific plant lectins commonly used for glycan blotting. Though a general reactivity towards glycoproteins might be expected from the avidity conferred by its high valence, it showed a marked preference for glycoproteins with multiple glycans, terminated by Gal or GalNAc. Thus its ten closely-spaced sites each with a K(D) for GalNAc of ~2 mM appeared to make RSVL more selective than the four more widely-spaced sites of soybean agglutinin, with a ten-fold better K(D) for GalNAc.Peer reviewed: YesNRC publication: Ye

Reducing the Stimulation of CD8+ T Cells during Infection with Intracellular Bacteria Promotes Differentiation Primarily into a Central (CD62LhighCD44high) Subset.

NRC publication: Ye

Monoclonal antibodies recognizing the surface autolysin IspC of Listeria monocytogenes serotype 4b: epitope localization, kinetic characterization, and cross-reaction studies.

Listeria monocytogenes serotype 4b is responsible for a high percentage of fatal cases of food-borne infection. In a previous study, we created 15 monoclonal antibodies (MAbs) against a ≈ 77 kDa antigen that is associated with the cell surface of live L. monocytogenes serotype 4b cells. Here we report an extensive characterization of these MAbs to further their development as diagnostic reagents. The ≈ 77 kDa target antigen was identified by mass spectrometry and N-terminal sequencing to be IspC, a novel surface associated autolysin. Epitope localization experiments revealed that each of the 15 MAbs recognized the C-terminal cell-wall binding domain of IspC. The presence of IspC was shown to be highly conserved within L. monocytogenes serotype 4b, as evidenced by a strong reaction between anti-IspC MAbs and all 4b isolates. To determine the range of cross-reactivity with other L. monocytogenes serotypes ELISA was used to test each MAb against multiple isolates from each of the L. monocytogenes serotypes. Of the 15 MAbs, five: M2774, M2775, M2780, M2790 and M2797, showed specificity for L. monocytogenes serotype 4b and only cross reacted with serotype 4ab isolates. The kinetics of the interaction between each of the MAbs and IspC was measured using surface plasmon resonance. The MAbs M2773, M2792, M2775, M2797 and M2781 each had very low dissociation constants (4.5 × 10(-9) to 1.2 × 10(-8) M). While several of these antibodies have properties which could be useful in diagnostic tests, the combined high fidelity and affinity of M2775 for the IspC protein and serotype 4b isolates, makes it a particularly promising candidate for use in the development of a specific L. monocytogenes serotype 4b diagnostic test

Neutrophils Play an Important Role in Host Resistance to Respiratory Infection with Acinetobacter baumannii in Mice▿

Acinetobacter baumannii has emerged as a major cause of both community-associated and nosocomial pneumonia, but little is known about the cellular and molecular mechanisms of host defense against respiratory infection with this bacterial pathogen. In this study, we examined the role of neutrophils in host resistance to pulmonary A. baumannii infection in a mouse model of intranasal (i.n.) infection. We found that neutrophils were rapidly recruited to the lungs following i.n. inoculation of the pathogen and declined to baseline level upon clearance of the infection. Depletion of neutrophils using monoclonal antibody RB6-8C5 prior to infection resulted in an acute lethal infection that was associated with enhanced bacterial burdens in the lung (P < 0.05) and extrapulmonary dissemination to the spleen. The increased susceptibility to A. baumannii in neutropenic mice was associated with a delay in the mRNA expression and production of early proinflammatory cytokines such as tumor necrosis factor alpha, interleukin-6, keratinocyte chemoattractant protein, monocyte chemoattractant protein 1, and macrophage inflammatory protein 2 (MIP-2) in the lungs and development of severe bronchopneumonia and lymphoid tissue destruction in the spleen. Moreover, i.n. administration of the neutrophil-inducing chemokine MIP-2 to normal mice induced a pulmonary influx of neutrophils and significantly enhanced the clearance of A. baumannii from the lungs (P < 0.01). These results imply that neutrophils play a critical role in host resistance to respiratory A. baumannii infection