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During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin-ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (VHHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such VHHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these VHHs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as "intrabodies," these VHHs rendered cells resistant to ricin intoxication. One VHH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation. [ABSTRACT FROM AUTHOR]

Camelid-derived and synthetic single-domain antibodies (sdAbs) are emerging as potent weapons against the novel coronavirus, SARS-CoV-2. sdAbs are small, compact, thermostable immunoglobulin elements capable of binding targets with subnanomolar affinities. By leveraging the power of phage- and yeast surface-display technologies, rare sdAbs can be isolated from highly diverse and complex antibody libraries. Once in hand, sdAbs can be engineered to improve binding affinity, avidity, target specificities, and biodistribution. In this Opinion piece we highlight a series of sophisticated studies describing the identification of ultrapotent sdAbs directed against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. We discuss the possible applications of these antibodies in the global fight against COVID-19. sdAbs are small, thermostable immunoglobulin elements that were first discovered in camels. Recent reports describe the isolation of sdAbs specific to the novel coronavirus, SARS-CoV-2, using phage- and yeast-display libraries. sdAbs with SARS-CoV-2 neutralizing activity target the trimeric Spike protein and block entry of the virus into host cells. Ultrapotent sdAbs have broad applications as antivirals and diagnostics in the fight against COVID-19. [ABSTRACT FROM AUTHOR]

Ricin toxin is a heterodimer consisting of RTA, a ribosomeinactivating protein, and RTB, a lectin that facilitates receptormediated uptake into mammalian cells. In previous studies, we demonstrated that toxin-neutralizing antibodies target four spatially distinct hot spots on RTA, which we refer to as epitope clusters I-IV. In this report, we identified and characterized three single domain camelid antibodies (VHH) against cluster II. One of these VHHs, V5E1, ranks as one of the most potent ricinneutralizing antibodies described to date. We solved the X-ray crystal structures of each of the three VHHs (E1, V1C7, and V5E1) in complex with RTA. V5E1 buries a total of 1,133 Ų of surface area on RTA and makes primary contacts with α-helixA (residues 18-32), α-helix F (182-194), as well as the F-G loop. V5E1, by virtue of complementarity determining region 3 (CDR3), may also engage with RTB and potentially interfere with the high affinity galactose-recognition element that plays a critical role in toxin attachment to cell surfaces and intracellular trafficking. The two other VHHs, E1 and V1C7, bind epitopes adjacent to V5E1 but display only weak toxin neutralizing activity, thereby providing structural insights into specific residues within cluster II that may be critical contact points for toxin inactivation. [ABSTRACT FROM AUTHOR]

During transport and storage, vaccines may be exposed to temperatures outside of the range recommended for storage, potentially causing efficacy losses. To better understand and prevent such losses, dominant negative inhibitor ( DNI), a recombinant protein antigen for a candidate vaccine against anthrax, was formulated as a liquid and as a glassy lyophilized powder with the adjuvants aluminum hydroxide and glycopyranoside lipid A ( GLA). Freeze-thawing of the liquid vaccine caused the adjuvants to aggregate and decreased its immunogenicity in mice. Immunogenicity of liquid vaccines also decreased when stored at 40°C for 8 weeks, as measured by decreases in neutralizing antibody titers in vaccinated mice. Concomitant with efficacy losses at elevated temperatures, changes in DNI structure were detected by fluorescence spectroscopy and increased deamidation was observed by capillary isoelectric focusing (cIEF) after only 1 week of storage of the liquid formulation at 40°C. In contrast, upon lyophilization, no additional deamidation after 4 weeks at 40°C and no detectable changes in DNI structure or reduction in immunogenicity after 16 weeks at 40°C were observed. Vaccines containing aluminum hydroxide and GLA elicited higher immune responses than vaccines adjuvanted with only aluminum hydroxide, with more mice responding to a single dose. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:627-639, 2015 [ABSTRACT FROM AUTHOR]

In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population. [ABSTRACT FROM AUTHOR]

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH "heterodimers." As compared with equimolar concentrations of their respective monovalent monomers, all three VHHheterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 x LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Residues Y91-T116 of ricin toxin''s enzymatic subunit (RTA) constitute an immunodominant loop-helix-loop motif that is the target of two potent toxin neutralizing monoclonal antibodies (mAbs), PB10 and R70. To define the exact epitope(s) recognized by these mAbs, we affinity enriched from a phage-displayed peptide library 12 mers that bound one or both of these mAbs. We report that PB10 recognizes a distinct but overlapping epitope with R70, in which residues Q98, E102, T105, and H106 are central to mAb recognition. [Copyright &y& Elsevier]

Abstract: The seasonal life-history patterns of penaeid prawns are complex and there are distinct differences between species. Better knowledge and understanding of offshore migration to the fishing grounds of penaeid prawns is particularly important for the operation and management of the Northern Prawn Fishery, Australia. Knowledge of the location of prawns during the critical spawning period of the year is important, especially since recent work suggests that inshore areas are critical for effective spawning. This paper explores factors, such as rainfall, water depth and season, impacting prawn migratory patterns, and models the spatial and temporal distribution of three commercially valuable species of prawns (Penaeus merguiensis, Penaeus semisulcatus and Penaeus esculentus) in Albatross Bay, Karumba and Mornington Island in the Northern Prawn Fishery using modern statistical methodology. The methodology allows for flexible functional relationships between variables, including univariate smooth terms and smooth terms for interactions between covariates: a major advancement on the methodology used in previous analyses of spatial distribution of prawns in the Gulf of Carpentaria. The models interpolate well, but expose the problems associated with extrapolation beyond the data. The results suggest that there are spawners in the important inshore waters during the critical spawning period of the year, although there is no strong evidence for major congregations of prawns in these important inshore regions compared to more offshore regions. Rainfall was significant in many of the models, particularly those describing the distribution of Penaeus merguiensis. Increased rainfall mostly had a positive effect on prawn catch rates, possibly by stimulating prawns to move out of shallow coastal areas into the fishing area. However, in some cases, high levels of rainfall had a negative effect which may be due to storms associated with the heavy rainfall decreasing the effectiveness of the fishing fleet. The analyses showed no signs of major inshore migrations associated with spawning, but there were certainly some spawners close inshore at the critical spawning period. [Copyright &y& Elsevier]