Satoru Horiya

Recent strategies targeting HIV glycans in vaccine design

Although efforts to develop a vaccine against HIV have so far met with little success, recent studies of HIV-positive patients with strongly neutralizing sera have shown that the human immune system is capable of producing potent and broadly neutralizing antibodies (bnAbs), some of which neutralize up to 90% of HIV strains. These antibodies bind conserved vulnerable sites on the viral envelope glycoprotein gp120, and identification of these sites has provided exciting clues about the design of potentially effective vaccines. Carbohydrates have a key role in this field, as a large fraction of bnAbs bind carbohydrates or combinations of carbohydrate and peptide elements on gp120. Additionally, carbohydrates partially mask some peptide surfaces recognized by bnAbs. The use of engineered glycoproteins and other glycostructures as vaccines to elicit antibodies with broad neutralizing activity is therefore a key area of interest in HIV vaccine design.

Directed Evolution of Multivalent Glycopeptides Tightly Recognized by HIV Antibody 2G12

Herein, we report a method for in vitro selection of multivalent glycopeptides, combining mRNA display with incorporation of unnatural amino acids and “click” chemistry. We have demonstrated the use of this method to design potential glycopeptide vaccines against HIV. From libraries of ∼1013 glycopeptides containing multiple Man9 glycan(s), we selected variants that bind to HIV broadly neutralizing antibody 2G12 with picomolar to low nanomolar affinity. This is comparable to the strength of the natural 2G12–gp120 interaction, and is the strongest affinity achieved to date with constructs containing 3–5 glycans. These glycopeptides are therefore of great interest in HIV vaccine design.

Replacement of the λ boxB RNA–N peptide with heterologous RNA–peptide interactions relaxes the strict spatial requirements for the formation of a transcription anti‐termination complex

In bacteriophage λ, formation of a transcriptional anti‐termination complex involving the elongating RNA polymerase is mediated by the interaction of boxB RNA with the RNA‐binding domain of the N protein (N peptide). In an attempt to understand the spatial requirements for boxB/N peptide interaction within the anti‐termination complex, the effects of changes in the distance between boxA and boxB RNA, the length of the boxB stem, and the distance between the N peptide and remainder of the N protein were examined using a bacterial reporter system. It was found that the requirements for boxB stem length and the distance between N peptide and the remainder of N were optimized and strict. In contrast, replacement of the boxB/N interaction by heterologous RNA–peptide interactions appeared to relax the strict requirement for RNA stem length and the orientation of the RNA‐binding peptide, presumably due to the absence of the cooperative interaction between boxB/N and the host factor NusA. In addition, the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer. A further understanding of the structural organization of the anti‐termination complex may provide insights into how functional ribonucleoprotein complexes may be engineered.

Synthesis of multivalent glycopeptide conjugates that mimic an HIV epitope

Recently, we reported a directed evolution method which enabled us to discover sequences of glycopeptides that bind with picomolar affinity to HIV antibody 2G12 and are of interest as HIV vaccine candidates. In this manuscript, we describe the syntheses of several of these large (~11-12 kDa) glycopeptides by a combination of fast flow peptide synthesis and click chemistry. We also discuss the optimization of their attachment to carrier protein CRM197, affording antigenic and immunogenic conjugates ready for animal vaccination.

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.

Directed Evolution of Glycopeptides Using mRNA Display

Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein. Although yeast, mammalian and Escherichia coli cells can be engineered to produce a homogenous glycoform at all glycosylation sites, there are just a few specific glycan structures that can readily be accessed in this manner. Recently, we reported a novel system for the directed evolution of glycopeptide libraries, which could in principle be decorated with any desired glycan. Our method combines in vitro peptide selection by mRNA display with unnatural amino acid incorporation and chemical attachment of synthetic oligosaccharides. Here, we provide an updated and optimized protocol for this method, which is designed to create glycopeptide mRNA display libraries containing ~1013 sequences and select them for target binding. The target described here is the HIV broadly neutralizing monoclonal antibody 2G12; 2G12 binds to cluster of high-mannose oligosaccharides on the HIV envelope glycoprotein gp120; and glycopeptides that mimic this epitope may be useful in HIV vaccine applications. This method is expected to be readily applicable for other types of glycans and targets of interest in glycobiology.

Analysis of the spacial requirements for RNA-protein interactions within the N antitermination complex of bacteriophage λ

In bacteriophage lambda, formation of a transcriptional antitermination complex consisting of the lambda N protein, nut RNA transcript (boxA-boxB), host factors, and RNA polymerase is mediated by the interaction of the boxB RNA with the RNA-binding domain of N. In order to understand the spacial requirements of this boxB/N interaction within the complex, the effects of changes in the length of the nut site linker, the boxB stem, and the peptide spacer connecting the RNA-binding domain and activation domain of N were examined using a bacterial reporter system. As a result, we found that the requirements for the boxB stem length and N peptide linker length were optimized and strict. In contrast, when the boxB/N interaction was replaced by heterologous RNA/peptide interactions, the strict requirement for the length of the peptide linker and the RNA stem was relaxed, presumably due to the absence of the interaction between boxB/N and the host factor NusA in the wild-type complex. It was also shown that the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer, suggesting that a further understanding of the organization of the antitermination complex may provide insights into the engineering of functional ribonucleoprotein complexes.

Analysis of the interaction between selected RNA-binding peptides and a target RNA containing a bulge and a GNRA-type tetraloop

We have characterized the interaction between selected novel RNA-binding peptides and their target RNA. The RNA is comprised of two elements, a GCAA tetraloop, a member of the thermodynamically stable GNRA-type (where N is A or G, U, C; R is G or A) tetraloops, and a tri-purine bulge found in the frameshift stimutating structure on the human immunodeficiency virus type 1 (HIV-1) gag-pol mRNA. Peptides that bind specifically to the target RNA were selected from a combinatorial library based on arginine-rich motif (ARM) by a bacterial reporter system. We performed mutational studies using the reporter system and gel shift assays and found that the binding affinity and specificity of the RNA were mainly dependent on the GNRA-type tetraloop, and a modest contribution was also attributed to the bulge structure. Our finding reveals a novel mode of interaction by an RNA-peptide complex and expands our knowledge on the diversity of molecular recognition.