Functional Stability of Unliganded Envelope Glycoprotein Spikes among Isolates of Human Immunodeficiency Virus Type 1 (HIV-1)

The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T90 (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines

Opportunities and challenges for monitoring terrestrial biodiversity in the robotics age

With biodiversity loss escalating globally, a step change is needed in our capacity to accurately monitor species populations across ecosystems. Robotic and autonomous systems (RAS) offer technological solutions that may substantially advance terrestrial biodiversity monitoring, but this potential is yet to be considered systematically. We used a modified Delphi technique to synthesize knowledge from 98 biodiversity experts and 31 RAS experts, who identified the major methodological barriers that currently hinder monitoring, and explored the opportunities and challenges that RAS offer in overcoming these barriers. Biodiversity experts identified four barrier categories: site access, species and individual identification, data handling and storage, and power and network availability. Robotics experts highlighted technologies that could overcome these barriers and identified the developments needed to facilitate RAS-based autonomous biodiversity monitoring. Some existing RAS could be optimized relatively easily to survey species but would require development to be suitable for monitoring of more ‘difficult’ taxa and robust enough to work under uncontrolled conditions within ecosystems. Other nascent technologies (for instance, new sensors and biodegradable robots) need accelerated research. Overall, it was felt that RAS could lead to major progress in monitoring of terrestrial biodiversity by supplementing rather than supplanting existing methods. Transdisciplinarity needs to be fostered between biodiversity and RAS experts so that future ideas and technologies can be codeveloped effectively

Opportunities and challenges for monitoring terrestrial biodiversity in the robotics age

With biodiversity loss escalating globally, a step-change is needed in our capacity to accurately monitor species populations across ecosystems. Robotic and autonomous systems (RAS) offer technological solutions that may significantly advance terrestrial biodiversity monitoring, but this potential is yet to be considered systematically. We used a modified Delphi technique to synthesise knowledge from 98 biodiversity and 31 RAS experts who identified the major methodological barriers that currently hinder monitoring, and explored the opportunities and challenges that RAS offer to overcome these barriers. Biodiversity experts identified four barrier categories: site access, species/individual identification, data handling/storage and power/network availability. Robotics experts highlighted technologies that could overcome these barriers and identified the developments needed to facilitate RAS-based autonomous biodiversity monitoring. Some existing RAS could be optimised relatively easily to survey species, but would require development to monitor more ‘difficult’ taxa and be robust enough to work in uncontrolled conditions within ecosystems. Other nascent technologies (e.g., novel sensors, biodegradable robots) need accelerated research. Overall, it was felt that RAS could lead to major progress in monitoring terrestrial biodiversity by supplementing, rather than supplanting, existing methods. Transdisciplinarity needs to be fostered between biodiversity and RAS experts, so future ideas and technologies can be co-developed effectively

N-linked glycosylation of HIV-1 and Hantaan virus glycoproteins. Implications for vaccination and therapy

Enveloped viruses have a great advantage comparedto the non-enveloped viruses due to theirability to acquire not only a host membrane butalso host-specific glycosylation of their surfaceglycoproteins. These carbohydrate structures,mainly of the N-linked glycan class, have a lowimmunogenicity and this feature enables envelopedviruses to keep a low profile towards theimmune system by shielding crucial epitopesfrom antibody recognition. This phenomenonconstitutes hurdles for development of vaccinesand therapies targeting highly glycosylated virusesincluding HIV and Hantaan virus. The aimsof the present work were to identify specific Nlinkedglycans that are especially active in thisvirus-shielding process and develop DNA vaccinesand therapeutic vectors to the two virusesmentioned.An N-linked glycan attached to N306 in the V3loop of the HIV-1 gp120 is involved in protectingthe virus from antibodies. A DNA vaccinewas created, where this particular glycosylationsite was eliminated using site-directed mutagenesis.Mice were immunised intranasally withthe mutant and a wild type vaccine, and theimmune responses were analysed. The protocolincluded an analysis of animals immunised withDNA only and also those given a protein boost.The mutant DNA followed by protein boostinduced a significantly higher IgG response togp120 as measured by ELISA. Both DNA vaccinesinduced similar levels of HIV-neutralisingantibodies in serum. The IgA response was analysedboth for BAL and faeces specimens. SecretedIgA was induced by both DNA vaccinesbut protein boost was required to obtain virusneutralisingantibodies.Attempts were made to construct an adenovirusvector, with receptor specificity retargeted togp120 of the HIV-infected cell surface. To obtaina suitable affinity module for gp120 a combinatorialprotein library of 108 members, based onthe 58 amino acid residue staphylococcal proteinA (Affibody), was screened against gp120.The selected affibody (zg120) and its bivalentform had Kd- values of 100 nM and 10 nM,respectively, towards gp120. The affibody constructswere introduced to the adenovirus 5 fibregene, and the recombinant fibres bound selectivelyto gp120 in a biosensor analysis and togp160, transiently expressed in GMK cells. Thesuccessful selection of a gp120-binding affibodyligand indicates that future affibody-basedstrategies might evolve to complement antibodybasedefforts for HIV-1 therapy.The same strategy as described above for HIV-1was also applied to develop a DNA vaccine toHantaan virus glycoproteins. All six potentialglycosylation sites (N1-N6) of the two Hantaanvirus glycoproteins G1 and G2 were eliminatedand expressed as single mutations in a DNAvaccine. Mice were immunized by Gene Gunwith these constructs. One mutated DNA vaccineconstruct, N6, was found to induce equallyhigh neutralisation titres as did the wild typeDNA vaccine. However, the mutant but not thewild type DNA vaccine was able to induce partialprotection against Hantaan virus challenge.In conclusion, manipulation of glycosylationsites of viral glycoprotein genes is still open as aroad to an improved DNA vaccine capable ofeliciting high titres of broadly neutralising antibodies,but it is clear that there are still obstaclesto be overcome

Demonstration of neutralizing mucosal IgA response to intranasal HIV-1 env DNA vaccines with or without the V3 glycosylation site

HIV-1 env based DNA vaccines are generally found to be poor B-cell immunogens. We examined the role of an N-glycan located in the V3 loop of HIV-1 (N306) that is known to modulate the immunogenicity of gp120. Here we describe intranasal immunizations with env (HIV-1 BRU) based genetic immunogens in combination with subcutaneous boosts of recombinant gp160 (rgp160) in mice. Immunization with DNA alone resulted in detectable IgA responses to rgp160 in both faeces and bronchoalveolar lavage (BAL) fluid, but the additional boosting increased the faecal IgA titres only. Protein boosting was required for induction of faecal IgA antibodies capable of neutralizing a homologous laboratory strain and a subtype B primary isolate. The B-cell response towards V3 loop peptides was not only directed against the homologous subtype B but also against the subtype F. In contrast to our previous observations on IgG, there were no differences in anti-gp160 IgA titres elicited by the N-glycan mutant and the wild-type immunogen. These results indicate that intranasal administration of plasmids containing env in combination with a subcutaneous boost proved to be an effective way of eliciting neutralizing mucosal IgA against HIV-1

Total Synthesis, Revised Structure, and Biological Evaluation of Biyouyanagin A and Analogues Thereof

Isolated from Hypericum species H. chinese L. var. salicifolium, biyouyanagin A was assigned structure 1a or 1b on the basis of NMR spectroscopic analysis. This novel natural product exhibited significant anti-HIV properties and inhibition of lipopolysaccharide-induced cytokine production. Described herein are the total syntheses of biyouyanagin A and several analogues (3−11), structural revision of biyouyanagin A to 2b, and the biological properties of all synthesized compounds. The total synthesis proceeded through cascade sequences that efficiently produced enantiomerically pure key building blocks 15b (ent-zingiberene) and 18 (hyperolactone C) and featured a novel [2 + 2] photoinduced cycloaddition reaction which occurred with complete regio- and stereoselectivity. Biological investigations with the synthesized biyouyangagins A (2−11) and hyperolactones C (12−16) revealed that the activity of biyouyanagin A most likely resides in its hyperolactone C structural domain

Total Synthesis, Revised Structure, and Biological Evaluation of Biyouyanagin A and Analogues Thereof

Isolated from Hypericum species H. chinese L. var. salicifolium, biyouyanagin A was assigned structure 1a or 1b on the basis of NMR spectroscopic analysis. This novel natural product exhibited significant anti-HIV properties and inhibition of lipopolysaccharide-induced cytokine production. Described herein are the total syntheses of biyouyanagin A and several analogues (3−11), structural revision of biyouyanagin A to 2b, and the biological properties of all synthesized compounds. The total synthesis proceeded through cascade sequences that efficiently produced enantiomerically pure key building blocks 15b (ent-zingiberene) and 18 (hyperolactone C) and featured a novel [2 + 2] photoinduced cycloaddition reaction which occurred with complete regio- and stereoselectivity. Biological investigations with the synthesized biyouyangagins A (2−11) and hyperolactones C (12−16) revealed that the activity of biyouyanagin A most likely resides in its hyperolactone C structural domain

Total Synthesis, Revised Structure, and Biological Evaluation of Biyouyanagin A and Analogues Thereof

Isolated from Hypericum species H. chinese L. var. salicifolium, biyouyanagin A was assigned structure 1a or 1b on the basis of NMR spectroscopic analysis. This novel natural product exhibited significant anti-HIV properties and inhibition of lipopolysaccharide-induced cytokine production. Described herein are the total syntheses of biyouyanagin A and several analogues (3−11), structural revision of biyouyanagin A to 2b, and the biological properties of all synthesized compounds. The total synthesis proceeded through cascade sequences that efficiently produced enantiomerically pure key building blocks 15b (ent-zingiberene) and 18 (hyperolactone C) and featured a novel [2 + 2] photoinduced cycloaddition reaction which occurred with complete regio- and stereoselectivity. Biological investigations with the synthesized biyouyangagins A (2−11) and hyperolactones C (12−16) revealed that the activity of biyouyanagin A most likely resides in its hyperolactone C structural domain

Total Synthesis, Revised Structure, and Biological Evaluation of Biyouyanagin A and Analogues Thereof

Isolated from Hypericum species H. chinese L. var. salicifolium, biyouyanagin A was assigned structure 1a or 1b on the basis of NMR spectroscopic analysis. This novel natural product exhibited significant anti-HIV properties and inhibition of lipopolysaccharide-induced cytokine production. Described herein are the total syntheses of biyouyanagin A and several analogues (3−11), structural revision of biyouyanagin A to 2b, and the biological properties of all synthesized compounds. The total synthesis proceeded through cascade sequences that efficiently produced enantiomerically pure key building blocks 15b (ent-zingiberene) and 18 (hyperolactone C) and featured a novel [2 + 2] photoinduced cycloaddition reaction which occurred with complete regio- and stereoselectivity. Biological investigations with the synthesized biyouyangagins A (2−11) and hyperolactones C (12−16) revealed that the activity of biyouyanagin A most likely resides in its hyperolactone C structural domain