Resurrected Ancestors Reveal Origins of Metamorphism in XCL1.

In a recent study, Dishman et al. resurrected ancestors of the metamorphic chemokine, XCL1, inferred through phylogenetics, and found that metamorphism arose in the XCL1 lineage ~150 million years ago. A zigzagging evolutionary path suggests that the metamorphic properties are adaptive and reveals three design principles that could be used for technological applications.

Efficient production of fluorophore-labeled CC chemokines for biophysical studies using recombinant enterokinase and recombinant sortase.

Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti-inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti-chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion-tagged chemokine is produced in Escherichia coli, then efficient cleavage of the N-terminal fusion partner is carried out with lab-produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab-produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP-fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti-inflammatory therapeutic, showing a binding constant for vCCI:vMIP-fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP-fluor) can be used in competition assays with other chemokines and we report a Kd for vCCI:CCL17 of 14 µM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations.

Griffithsin Retains Anti-HIV-1 Potency with Changes in gp120 Glycosylation and Complements Broadly Neutralizing Antibodies PGT121 and PGT126.

Griffithsin (Grft) is an antiviral lectin that has been shown to potently inhibit HIV-1 by binding high-mannose N-linked glycosylation sites on HIV-1 gp120. A key factor for Grft potency is glycosylation at N295 of gp120, which is directly adjacent to N332, a target glycan for an entire class of broadly neutralizing antibodies (bNAbs). Here, we unify previous work on the importance of other glycans to Grft potency against HIV-1 and Grft's role in mediating the conformational change of gp120 by mutating nearly every glycosylation site in gp120. In addition to a significant loss of Grft activity by the removal of glycosylation at N295, glycan absence at N332 or N448 was found to have moderate effects on Grft potency. Interestingly, in the absence of N295, Grft effectiveness could be improved by a mutation that results in the glycan at N448 shifting to N446, indicating that the importance of individual glycans may be related to their effect on glycosylation density. Grft's ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in clade B strains, not clade C strains. We further demonstrate that Grft can rescue the activity of the bNAbs PGT121 and PGT126 in the event of a loss or a shift of glycosylation at N332, where the bNAbs suffer a drastic loss of potency. Despite targeting the same region, Grft in combination with PGT121 and PGT126 produced additive effects. This indicates that Grft could be an important combinational therapeutic.

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1ß/CC-Chemokine Ligand 4 (MIP-1ß/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 µs molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1ß. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1ß, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1ß. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1ß interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.

Structural insights into the interaction between a potent anti-inflammatory protein, viral CC chemokine inhibitor (vCCI), and the human CC chemokine, Eotaxin-1.

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1ß (macrophage inflammatory protein-1ß) complex indicated that vCCI uses negatively charged residues in ß-sheet II to interact with positively charged residues in the MIP-1ß N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1ß complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1ß, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.

The griffithsin dimer is required for high-potency inhibition of HIV-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism.

Griffithsin (Grft) is a protein lectin derived from red algae that tightly binds the HIV envelope protein gp120 and effectively inhibits virus infection. This inhibition is due to the binding by Grft of high-mannose saccharides on the surface of gp120. Grft has been shown to be a tight dimer, but the role of the dimer in Grft's anti-HIV function has not been fully explored. To investigate the role of the Grft dimer in anti-HIV function, an obligate dimer of Grft was designed by expressing the protein with a peptide linker between the two subunits. This "Grft-linker-Grft" is a folded protein dimer, apparently nearly identical in structural properties to the wild-type protein. A "one-armed" obligate dimer was also designed (Grft-linker-Grft OneArm), with each of the three carbohydrate binding sites of one subunit mutated while the other subunit remained intact. While both constructed dimers retained the ability to bind gp120 and the viral surface, Grft-linker-Grft OneArm was 84- to 1,010-fold less able to inhibit HIV than wild-type Grft, while Grft-linker-Grft had near-wild-type antiviral potency. Furthermore, while the wild-type protein demonstrated the ability to alter the structure of gp120 by exposing the CD4 binding site, Grft-linker-Grft OneArm largely lost this ability. In experiments to investigate gp120 shedding, it was found that Grft has different effects on gp120 shedding for strains from subtype B and subtype C, and this might correlate with Grft function. Evidence is provided that the dimer form of Grft is critical to the function of this protein in HIV inhibition.

The Effect of Select SARS-CoV-2 N-Linked Glycan and Variant of Concern Spike Protein Mutations on C-Type Lectin-Receptor-Mediated Infection.

The SARS-CoV-2 virion has shown remarkable resilience, capable of mutating to escape immune detection and re-establishing infectious capabilities despite new vaccine rollouts. Therefore, there is a critical need to identify relatively immutable epitopes on the SARS-CoV-2 virion that are resistant to future mutations the virus may accumulate. While hACE2 has been identified as the receptor that mediates SARS-CoV-2 susceptibility, it is only modestly expressed in lung tissue. C-type lectin receptors like DC-SIGN can act as attachment sites to enhance SARS-CoV-2 infection of cells with moderate or low hACE2 expression. We developed an easy-to-implement assay system that allows for the testing of SARS-CoV-2 trans-infection. Using our assay, we assessed how SARS-CoV-2 Spike S1-domain glycans and spike proteins from different strains affected the ability of pseudotyped lentivirions to undergo DC-SIGN-mediated trans-infection. Through our experiments with seven glycan point mutants, two glycan cluster mutants and four strains of SARS-CoV-2 spike, we found that glycans N17 and N122 appear to have significant roles in maintaining COVID-19's infectious capabilities. We further found that the virus cannot retain infectivity upon the loss of multiple glycosylation sites, and that Omicron BA.2 pseudovirions may have an increased ability to bind to other non-lectin receptor proteins on the surface of cells. Taken together, our work opens the door to the development of new therapeutics that can target overlooked epitopes of the SARS-CoV-2 virion to prevent C-type lectin-receptor-mediated trans-infection in lung tissue.

The Antiviral Activity of the Lectin Griffithsin against SARS-CoV-2 Is Enhanced by the Presence of Structural Proteins.

Although COVID-19 transmission has been reduced by the advent of vaccinations and a variety of rapid monitoring techniques, the SARS-CoV-2 virus itself has shown a remarkable ability to mutate and persist. With this long track record of immune escape, researchers are still exploring prophylactic treatments to curtail future SARS-CoV-2 variants. Specifically, much focus has been placed on the antiviral lectin Griffithsin in preventing spike protein-mediated infection via the hACE2 receptor (direct infection). However, an oft-overlooked aspect of SARS-CoV-2 infection is viral capture by attachment receptors such as DC-SIGN, which is thought to facilitate the initial stages of COVID-19 infection in the lung tissue (called trans-infection). In addition, while immune escape is dictated by mutations in the spike protein, coronaviral virions also incorporate M, N, and E structural proteins within the particle. In this paper, we explored how several structural facets of both the SARS-CoV-2 virion and the antiviral lectin Griffithsin can affect and attenuate the infectivity of SARS-CoV-2 pseudovirus. We found that Griffithsin was a better inhibitor of hACE2-mediated direct infection when the coronaviral M protein is present compared to when it is absent (possibly providing an explanation regarding why Griffithsin shows better inhibition against authentic SARS-CoV-2 as opposed to pseudotyped viruses, which generally do not contain M) and that Griffithsin was not an effective inhibitor of DC-SIGN-mediated trans-infection. Furthermore, we found that DC-SIGN appeared to mediate trans-infection exclusively via binding to the SARS-CoV-2 spike protein, with no significant effect observed when other viral proteins (M, N, and/or E) were present. These results provide etiological data that may help to direct the development of novel antiviral treatments, either by leveraging Griffithsin binding to the M protein as a novel strategy to prevent SARS-CoV-2 infection or by narrowing efforts to inhibit trans-infection to focus on DC-SIGN binding to SARS-CoV-2 spike protein.

Sustained Delivery of the Antiviral Protein Griffithsin and Its Adhesion to a Biological Surface by a Silk Fibroin Scaffold.

The protein Griffithsin (Grft) is a lectin that tightly binds to high-mannose glycosylation sites on viral surfaces. This property allows Grft to potently inhibit many viruses, including HIV-1. The major route of HIV infection is through sexual activity, so an important tool for reducing the risk of infection would be a film that could be inserted vaginally or rectally to inhibit transmission of the virus. We have previously shown that silk fibroin can encapsulate, stabilize, and release various antiviral proteins, including Grft. However, for broad utility as a prevention method, it would be useful for an insertable film to adhere to the mucosal surface so that it remains for several days or weeks to provide longer-term protection from infection. We show here that silk fibroin can be formulated with adhesive properties using the nontoxic polymer hydroxypropyl methylcellulose (HPMC) and glycerol, and that the resulting silk scaffold can both adhere to biological surfaces and release Grft over the course of at least one week. This work advances the possible use of silk fibroin as an anti-viral insertable device to prevent infection by sexually transmitted viruses, including HIV-1.

Highly potent chimeric inhibitors targeting two steps of HIV cell entry.

Blocking HIV-1 cell entry has long been a major goal of anti-HIV drug development. Here, we report a successful design of two highly potent chimeric HIV entry inhibitors composed of one CCR5-targeting RANTES (regulated on activation normal T cell expressed and secreted) variant (5P12-RANTES or 5P14-RANTES (Gaertner, H., Cerini, F., Escola, J. M., Kuenzi, G., Melotti, A., Offord, R., Rossitto-Borlat, I., Nedellec, R., Salkowitz, J., Gorochov, G., Mosier, D., and Hartley, O. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 17706-17711)) linked to a gp41 fusion inhibitor, C37. Chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed extremely high antiviral potency in single cycle and replication-competent viral assays against R5-tropic viruses, with IC(50) values as low as 0.004 nm. This inhibition was somewhat strain-dependent and was up to 100-fold better than the RANTES variant alone or in combination with unlinked C37. The chimeric inhibitors also fully retained the antiviral activity of C37 against X4-tropic viruses, and this inhibition can be further enhanced significantly if the target cell co-expresses CCR5 receptor. On human peripheral blood mononuclear cells, the inhibitors showed very strong inhibition against R5-tropic Ba-L strain and X4-tropic IIIB strain, with IC(50) values as low as 0.015 and 0.44 nm, which are 45- and 16-fold better than the parent inhibitors, respectively. A clear delivery mechanism requiring a covalent linkage between the two segments of the chimera was observed and characterized. Furthermore, the two chimeric inhibitors are fully recombinant and are easily produced at low cost. These attributes make them excellent candidates for anti-HIV microbicides. The results of this study also suggest a potent approach for optimizing existing HIV entry inhibitors or designing new inhibitors.

The role of individual carbohydrate-binding sites in the function of the potent anti-HIV lectin griffithsin.

Griffithsin (GRFT) is a lectin that has been shown to inhibit HIV infection by binding to high mannose glycan structures on the surface of gp120, and it is among the most potent HIV entry inhibitors reported so far. However, important biochemical details on the antiviral mechanism of GRFT action remain unexplored. In order to understand the role of the three individual carbohydrate-binding sites (CBS) in GRFT, mutations were made at each site (D30A, D70A, and D112A), and the resulting mutants were investigated. NMR studies revealed that each GRFT variant was folded but showed significant peak movement on the carbohydrate-binding face of the protein. The wild-type and each point mutant protein appeared as tight dimers with a K(d) below 4.2 µM. Mutation of any individual CBS on GRFT reduced binding of the protein to mannose, and ELISA assays revealed a partial loss of ability of each GRFT point mutant to bind gp120, with a near-complete loss of binding by the triple mutant D30A/D70A/D112A GRFT. A more quantitative surface plasmon resonance (SPR) examination showed a rather small loss of binding to gp120 for the individual GRFT point mutants (K(D): 123 to 245 pM range versus 73 pM for wild-type GRFT), but dramatic loss of the triple mutant to bind gp120 derived from R5 and X4 strains (K(D) > 12 nM). In contrast to the 2- to 3-fold loss of binding to gp120, the single CBS point mutants of GRFT were significantly less able to inhibit viral infection, exhibiting a 26- to 1900-fold loss of potency, while the triple mutant was at least 875-fold less effective against HIV-1 infection. The disparity between HIV-1 gp120 binding ability and HIV inhibitory potency for these GRFT variants indicates that gp120 binding and virus neutralization do not necessarily correlate, and suggests a mechanism that is not based on simple gp120 binding.

The binding and specificity of chemokine binding proteins, through the lens of experiment and computation.

Chemokines are small proteins that are critical for immune function, being primarily responsible for the activation and chemotaxis of leukocytes. As such, many viruses, as well as parasitic arthropods, have evolved systems to counteract chemokine function in order to maintain virulence, such as binding chemokines, mimicking chemokines, or producing analogs of transmembrane chemokine receptors that strongly bind their targets. The focus of this review is the large group of chemokine binding proteins (CBP) with an emphasis on those produced by mammalian viruses. Because many chemokines mediate inflammation, these CBP could possibly be used pharmaceutically as anti-inflammatory agents. In this review, we summarize the structural properties of a diverse set of CBP and describe in detail the chemokine binding properties of the poxvirus-encoded CBP called vCCI (viral CC Chemokine Inhibitor). Finally, we describe the current and emerging capabilities of combining computational simulation, structural analysis, and biochemical/biophysical experimentation to understand, and possibly re-engineer, protein-protein interactions.

SARS-CoV-2 pseudotyped virus persists on the surface of multiple produce but can be inactivated with gaseous ozone.

Due to the immense societal and economic impact that the COVID-19 pandemic has caused, limiting the spread of SARS-CoV-2 is one of the most important priorities at this time. The global interconnectedness of the food industry makes it one of the biggest concerns for SARS-CoV-2 outbreaks. Although fomites are currently considered a low-risk route of transmission for SARS-CoV-2, new variants of the virus can potentially alter the transmission dynamics. In this study, we compared the survival rate of pseudotyped SARS-CoV-2 on plastic with some commonly used food samples (i.e., apple, strawberry, grapes, tomato, cucumber, lettuce, parsley, Brazil nut, almond, cashew, and hazelnut). The porosity level and the chemical composition of different food products affect the virus's stability and infectivity. Our results showed that tomato, cucumber, and apple offer a higher survival rate for the pseudotyped viruses. Next, we explored the effectiveness of ozone in deactivating the SARS-CoV-2 pseudotyped virus on the surface of tomato, cucumber, and apple. We found that the virus was effectively inactivated after being exposed to 15 ppm of ozone for 1 h under ambient conditions. SEM imaging revealed that while ozone exposure altered the wax layer on the surface of produce, it did not seem to damage the cells and their biological structures. The results of our study indicate that ozonated air can likely provide a convenient method of effectively disinfecting bulk food shipments that may harbour the SARS-CoV-2 virus.

Potent strategy to inhibit HIV-1 by binding both gp120 and gp41.

The development of an anti-HIV microbicide is critical in the fight against the spread of HIV. It is shown here that the covalent linking of compounds that bind gp120 with compounds that bind gp41 can inhibit HIV entry even more potently than individual inhibitors or noncovalent combinations. The most striking example involves griffithsin, a potent HIV inhibitor that binds to the surface of HIV gp120. While griffithsin inhibits HIV Env-mediated fusion in a CCR5-tropic cell-cell fusion assay with a 50% inhibitory concentration (IC(50)) of 1.31 ± 0.87 nM and the gp41-binding peptide C37 shows an IC(50) of 18.2 ± 7.6 nM, the covalently linked combination of griffithsin with C37 (Griff37) has an IC(50) of 0.15 ± 0.05 nM, exhibiting a potency 8.7-fold greater than that of griffithsin alone. Similarly, in CXCR4-tropic cell-cell fusion assays, Griff37 is 5.2-fold more potent than griffithsin alone. In viral assays, both griffithsin and Griff37 inhibit HIV replication at midpicomolar levels, but the linked compound Griff37 is severalfold more potent than griffithsin alone against both CCR5- and CXCR4-tropic virus strains. Another example of this strategy is the covalently linked combination of peptide C37 with a variant of the gp120-binding peptide CD4M33 (L. Martin et al., Nat. Biotechnol. 21:71-76, 2003). Also, nuclear magnetic resonance (NMR) spectra for several of these compounds are shown, including, to our knowledge, the first published NMR spectrum for griffithsin.

Characterization of the interactions of vMIP-II, and a dimeric variant of vMIP-II, with glycosaminoglycans.

Chemokines are important immune proteins, carrying out their function by binding to glycosaminoglycans (GAGs) on the endothelial surface and to cell surface chemokine receptors. A unique viral chemokine analogue, viral macrophage inflammatory protein-II (vMIP-II), encoded by human herpesvirus-8, has garnered interest because of its ability to bind to multiple chemokine receptors, including both HIV coreceptors. In addition, vMIP-II binds to cell surface GAGs much more tightly than most human chemokines, which may be the key to its anti-inflammatory function in vivo. The goal of this work was to determine the mechanism of binding of GAG by vMIP-II. The interaction of vMIP-II with a heparin-derived disaccharide was characterized using NMR. Important binding sites were further analyzed by mutagenesis studies, in which corresponding vMIP-II mutants were tested for GAG binding ability using heparin chromatography and NMR. We found that despite having many more basic residues than some chemokines, vMIP-II shares a characteristic binding site similar to that of its human analogues, utilizing basic residues R18, R46, and R48. Interestingly, a particular mutation (Leu13Phe) caused vMIP-II to form a pH-dependent CC chemokine-type dimer as determined by analytical ultracentrifugation and NMR. To the best of our knowledge, this is the first example of engineering a naturally predominantly monomeric chemokine into a dissociable dimer by a single mutation. This dimeric vMIP-II mutant binds to heparin much more tightly than wild-type vMIP-II and provides a new model for studying the relationship between chemokine quaternary structure and various aspects of function. Structural differences between monomeric and dimeric vMIP-II upon GAG binding were characterized by NMR and molecular docking.

Structural and functional studies of the potent anti-HIV chemokine variant P2-RANTES.

The N-terminal region of the chemokine RANTES is critical for its function. A synthesized N-terminally modified analog of RANTES, P2-RANTES, was discovered using a phage display selection against living CCR5-expressing cells, and has been reported to inhibit HIV-1 env-mediated cell-cell fusion at subnanomolar levels (Hartley et al. J Virol 2003;77:6637-6644). In the present study we produced this protein using E. coli overexpression and extensively studied its structure and function. The x-ray crystal structure of P2-RANTES was solved and refined at 1.7 A resolution. This protein was found to be predominantly a monomer in solution by analytical ultracentrifugation, but a tetramer in the crystal. In studies of glycosaminoglycan binding, P2-RANTES was found to be significantly less able to bind heparin than wild type RANTES. We also tested this protein for receptor internalization where it was shown to be functional, in cell-cell fusion assays where recombinant P2-RANTES was a potent fusion inhibitor (IC(50) = 2.4 +/- 0.8 nM), and in single round infection assays where P2-RANTES inhibited at subnanomolar levels. Further, in a modified fusion assay designed to test specificity of inhibition, P2-RANTES was also highly effective, with a 65-fold improvement over the fusion inhibitor C37, which is closely related to the clinically approved inhibitor T-20. These studies provide detailed structural and functional information for this novel N-terminally modified chemokine mutant. This information will be very useful in the development of more potent anti-HIV agents. PDB Accession Number: 2vxw.

Efficacy of silk fibroin biomaterial vehicle for in vivo mucosal delivery of Griffithsin and protection against HIV and SHIV infection ex vivo.

INTRODUCTION: The majority of new HIV infections occur through mucosal transmission. The availability of readily applicable and accessible platforms for anti-retroviral (ARV) delivery is critical for the prevention of HIV acquisition through sexual transmission in both women and men. There is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. The silk fibroin (SF) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. The objective of this study was to test safety and efficacy of SF for anti-HIV drug delivery to mucosal sites and for viral prevention. METHODS: We formulated a potent HIV inhibitor Griffithsin (Grft) in a mucoadhesive silk fibroin (SF) drug delivery platform and tested the application in a non-human primate model in vivo and a pre-clinical human cervical and colorectal tissue explant model. Both vaginal and rectal compartments were assessed in rhesus macaques (Mucaca mulatta) that received SF (n = 4), no SF (n = 7) and SF-Grft (n = 11). In this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo SHIV challenge. RESULTS: Effective Grft release and retention in mucosal tissues from the SF-Grft platform resulted in protection against HIV in human cervical and colorectal tissue as well as against SHIV challenge in both rhesus macaque vaginal and rectal tissues. Mucoadhesion of SF-Grft inserts did not cause any inflammatory responses or changes in local microbiota. CONCLUSIONS: We demonstrated that in vivo delivery of SF-Grft in rhesus macaques fully protects against SHIV challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. Our study provides support for the development of silk fibroin as a highly promising, user-friendly HIV prevention modality to address the global disparity in HIV infection.

WITHDRAWN: Very strong synergy between modified RANTES and gp41 binding peptides leads to potent anti-HIV-1 activity.

This article, published ahead of print on 28 July 2008, has been withdrawn by the authors. Although moderate synergy between P2-RANTES and C peptides can be observed with high statistical significance in cell fusion assays, this synergy was not able to be verified in HIV viral assays. The authors regret the overstatement of synergy and will revise the paper for publication at a later date.

Sustained release silk fibroin discs: Antibody and protein delivery for HIV prevention.

With almost 2 million new HIV infections worldwide each year, the prevention of HIV infection is critical for stopping the pandemic. The only approved form of pre-exposure prophylaxis is a costly daily pill, and it is recognized that several options will be needed to provide protection to the various affected communities around the world. In particular, many at-risk people would benefit from a prevention method that is simple to use and does not require medical intervention or a strict daily regimen. We show that silk fibroin protein can be formulated into insertable discs that encapsulate either an antibody (IgG) or the potent HIV inhibitor 5P12-RANTES. Several formulations were studied, including silk layering, water vapor annealing and methanol treatment to stabilize the protein cargo and impact the release kinetics over weeks. In the case of IgG, high concentrations were released over a short time using methanol treatment, with more sustained results with the use of water vapor annealing and layering during device fabrication. For 5P12-RANTES, sustained release was obtained for 31 days using water vapor annealing. Further, we show that the released inhibitor 5P12-RANTES was functional both in vitro and in ex vivo colorectal tissue. This work shows that silk fibroin discs can be developed into formidable tools to prevent HIV infection.

Conservation of unfavorable sequence motifs that contribute to the chemokine quaternary state.

In the chemokine family, we characterize two examples of evolutionarily conserved unfavorable sequence motifs that affect quaternary structure. In contrast to the straightforward action of favorable sequences, these unfavorable motifs produce interactions disfavoring one outcome to indirectly promote another one but should not be confused with the broad sampling produced by negative selection and/or design. To identify such motifs, we developed a statistically validated computational method combining structure and phylogeny. This approach was applied in an analysis of the alternate forms of homodimerization exhibited in the chemokine family. While the chemokine family exhibits the same tertiary fold, members of certain subfamilies, including CXCL8, form a homodimer across the beta1 strand whereas members of other subfamilies, including CCL4 and CCL2, form a homodimer on the opposite side of the chemokine fold. These alternate dimerization states suggest that CCL4 and CCL2 contain specific sequences that disfavor CXCL8 dimerization. Using our computational approach, we identified two evolutionarily conserved sequence motifs in the CC subfamilies: a drastic two-residue deletion (DeltaRV) and a simple point mutation (V27R). Cloned into the CXCL8 background, these two motifs were experimentally proven to confer a monomeric state. NMR analyses indicate that these variants are structured in solution and retain the chemokine fold. Structurally, the motifs retain a chemokine tertiary fold while introducing unfavorable quaternary interactions that inhibit CXCL8 dimerization. In demonstrating the success of our computational method, our results argue that these unfavorable motifs have been evolutionarily conserved to specifically disfavor one dimerization state and, as a result, indirectly contribute to favoring another.