RESUMEN

More than 3 years into the global pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant threat to public health. Immunities acquired from infection or current vaccines fail to provide long term protection against subsequent infections, mainly due to their fast-waning nature and the emergence of variants of concerns (VOCs) such as Omicron. To overcome these limitations, SARS-CoV-2 Spike protein receptor binding domain (RBD)-based epitopes are investigated as conjugates with a powerful carrier, the mutant bacteriophage Qß (mQß). The epitope design is critical to eliciting potent antibody responses with the full length RBD being superior to peptide and glycopeptide antigens. The full length RBD conjugated with mQß activates both humoral and cellular immune systems in vivo, inducing broad spectrum, persistent, and comprehensive immune responses effective against multiple VOCs including Delta and Omicron variants, rendering it a promising vaccine candidate.

Asunto(s)

Linfocitos B , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Linfocitos T , SARS-CoV-2/inmunología , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Linfocitos T/inmunología , Animales , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Linfocitos B/inmunología , Ratones , Humanos , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/química , Mutación , Femenino , Allolevivirus/inmunología , Allolevivirus/química , Anticuerpos Antivirales/inmunología , Dominios Proteicos , Ratones Endogámicos BALB C , Inmunidad Celular

RESUMEN

Bacteriophage fitness is determined by factors influencing both their replication within bacteria and their ability to maintain infectivity between infections. The latter becomes particularly crucial under adverse environmental conditions or when host density is low. In such scenarios, the damage experienced by viral particles could lead to the loss of infectivity, which might be mitigated if the virus undergoes evolutionary optimization through replication. In this study, we conducted an evolution experiment involving bacteriophage Qß, wherein it underwent 30 serial transfers, each involving a cycle of freezing and thawing followed by replication of the surviving viruses. Our findings show that Qß was capable of enhancing its resistance to this selective pressure through various adaptive pathways that did not impair the virus replicative capacity. Notably, these adaptations predominantly involved mutations located within genes encoding capsid proteins. The adapted populations exhibited higher resistance levels than individual viruses isolated from them, and the latter surpassed those observed in single mutants generated via site-directed mutagenesis. This suggests potential interactions among mutants and mutations. In conclusion, our study highlights the significant role of extracellular selective pressures in driving the evolution of phages, influencing both the genetic composition of their populations and their phenotypic properties.

Asunto(s)

Congelación , Mutación , Fagos ARN/genética , Fagos ARN/fisiología , Adaptación Fisiológica/genética , Evolución Molecular , Replicación Viral/genética , Proteínas de la Cápside/genética

RESUMEN

I. Watanabe et al. isolated approximately 30 strains of RNA phages from various parts of Japan. To isolate RNA phages, they assessed the infection specificity of male Escherichia coli and RNase sensitivity. They found that the isolated strains of RNA phages could be serologically separated into three groups. Furthermore, most of them were serologically related, and the antiphage rabbit serum prepared by one of these phages neutralized most of the other phages. The only serologically unrelated phage was the RNA phage Qß, which was isolated at the Institute for Virus Research, Kyoto University, in 1961.

Asunto(s)

Fagos ARN , Humanos , Masculino , Conejos , Animales , Escherichia coli/genética , Japón

RESUMEN

Single-stranded RNA bacteriophages (ssRNA phages) are small viruses with a compact genome (~3-4 kb) that infect gram-negative bacteria via retractile pili. These phages have been applied in various fields since their discovery approximately 60 years ago. To understand their biology, it is crucial to analyze the structure of mature virions. Cryo-electron microscopy (cryo-EM) has been employed to determine the structures of two ssRNA phages, MS2 and Qß. This chapter presents a method for purifying these two phages and their receptor, the F-pilus, to allow examination using cryo-EM.

Asunto(s)

Bacteriófagos , Microscopía por Crioelectrón , Bacteriófagos/genética , ARN Viral/genética , Fimbrias Bacterianas , Levivirus/genética

RESUMEN

Introduction: Host density is one of the main factors affecting the infective capacity of viruses. When host density is low, it is more difficult for the virus to find a susceptible cell, which increases its probability of being damaged by the physicochemical agents of the environment. Nevertheless, viruses can adapt to variations in host density through different strategies that depend on the particular characteristics of the life cycle of each virus. In a previous work, using the bacteriophage Qß as an experimental model, we found that when bacterial density was lower than optimal the virus increased its capacity to penetrate into the bacteria through a mutation in the minor capsid protein (A1) that is not described to interact with the cell receptor. Results: Here we show that the adaptive pathway followed by Qß in the face of similar variations in host density depends on environmental temperature. When the value for this parameter is lower than optimal (30°C), the mutation selected is the same as at the optimal temperature (37°C). However, when temperature increases to 43°C, the mutation selected is located in a different protein (A2), which is involved both in the interaction with the cell receptor and in the process of viral progeny release. The new mutation increases the entry of the phage into the bacteria at the three temperatures assayed. However, it also considerably increases the latent period at 30 and 37°C, which is probably the reason why it is not selected at these temperatures. Conclusion: The conclusion is that the adaptive strategies followed by bacteriophage Qß, and probably other viruses, in the face of variations in host density depend not only on their advantages at this selective pressure, but also on the fitness costs that particular mutations may present in function of the rest of environmental parameters that influence viral replication and stability.

RESUMEN

The COVID-19 pandemic caused by SARS-CoV-2 sparked intensive research into the development of effective vaccines, 50 of which have been approved thus far, including the novel mRNA-based vaccines developed by Pfizer and Moderna. Although limiting the severity of the disease, the mRNA-based vaccines presented drawbacks, such as the cold chain requirement. Moreover, antibody levels generated by these vaccines decline significantly after 6 months. These vaccines deliver mRNA encoding the full-length spike (S) glycoprotein of SARS-CoV-2, but must be updated as new strains and variants of concern emerge, creating a demand for adjusted formulations and booster campaigns. To overcome these challenges, we have developed COVID-19 vaccine candidates based on the highly conserved SARS CoV-2, 809-826 B-cell peptide epitope (denoted 826) conjugated to cowpea mosaic virus (CPMV) nanoparticles and bacteriophage Qß virus-like particles, both platforms have exceptional thermal stability and facilitate epitope delivery with inbuilt adjuvant activity. We evaluated two administration methods: subcutaneous injection and an implantable polymeric scaffold. Mice received a prime-boost regimen of 100 µg per dose (2 weeks apart) or a single dose of 200 µg administered as a liquid formulation, or a polymer implant. Antibody titers were evaluated longitudinally over 50 weeks. The vaccine candidates generally elicited an early Th2-biased immune response, which stimulates the production of SARS-CoV-2 neutralizing antibodies, followed by a switch to a Th1-biased response for most formulations. Exceptionally, vaccine candidate 826-CPMV (administered as prime-boost, soluble injection) elicited a balanced Th1/Th2 immune response, which is necessary to prevent pulmonary immunopathology associated with Th2 bias extremes. While the Qß-based vaccine elicited overall higher antibody titers, the CPMV-induced antibodies had higher avidity. Regardless of the administration route and formulation, our vaccine candidates maintained high antibody titers for more than 50 weeks, confirming a potent and durable immune response against SARS-CoV-2 even after a single dose.

RESUMEN

GntR10 is a transcriptional regulator in Brucella. Nuclear factor-kappa B (NF-κB) is involved in many cellular activities, playing major roles in orchestrating the expression of inflammatory genes and regulating protein function that is essential for pathogenic bacteria during infection. GntR10 deletion was previously found to affect the growth and the virulence of Brucella and expression levels of target genes of GntR10 in mice. However, the mechanisms of affection of NF-κB regulated by Brucella GntR10 are still unclear. Here, GntR10 deletion could regulate the expression of LuxR-type transcriptional activators (VjbR and BlxR) of the quorum sensing system (QSS) and type IV secretion system (T4SS) effectors (BspE and BspF) of Brucella. It could further inhibit the activation of the regulator NF-κB and affect the virulence of Brucella. This research provides new insights into the designing of Brucella vaccines and the screening of drug targets. SIGNIFICANCE: Transcriptional regulators are predominant bacterial signal transduction factors. The pathogenicity of Brucella is due to its ability to regulate the expression of virulence related genes including quorum sensing system (QSS) and type IV secretion system (T4SS). Transcriptional regulators are designed to regulate gene expression and enact an appropriate adaptive physiological response. Here, we show that Brucella transcriptional regulator GntR10 regulated the expression of QSS and T4SS effectors, which affected the activation of NF-κB.

Asunto(s)

Brucella , Ratones , Animales , Brucella/metabolismo , FN-kappa B/metabolismo , Sistemas de Secreción Tipo IV/metabolismo , Percepción de Quorum/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Bacterianas/metabolismo

RESUMEN

How the ribonucleic acid (RNA) world transited to the deoxyribonucleic acid (DNA) world has remained controversial in evolutionary biology. At a certain time point in the transition from the RNA world to the DNA world, 'RNA replicons', in which RNAs produce proteins to replicate their coding RNA, and 'DNA replicons', in which DNAs produce RNA to synthesize proteins that replicate their coding DNA, can be assumed to coexist. The coexistent state of RNA replicons and DNA replicons is desired for experimental approaches to determine how the DNA world overtook the RNA world. We constructed a mini-RNA replicon in Escherichia coli. This mini-RNA replicon encoded the ß subunit, one of the subunits of the Qß replicase derived from the positive-sense single-stranded Qß RNA phage and is replicated by the replicase in E. coli. To maintain the mini-RNA replicon persistently in E. coli cells, we employed a system of α complementation of LacZ that was dependent on the Qß replicase, allowing the cells carrying the RNA replicon to grow in the lactose minimal medium selectively. The coexistent state of the mini-RNA replicon and DNA replicon (E. coli genome) was successively synthesized. The coexistent state can be used as a starting system to experimentally demonstrate the transition from the RNA-protein world to the DNA world, which will contribute to progress in the research field of the origin of life.

RESUMEN

Virus-like particles (VLPs) are promising scaffolds for biomaterials as well as diagnostic and therapeutic applications. However, there are some key challenges to be solved, such as the ability to engineer alternate sizes for varied use cases. To this end, we created a library of MS2 VLP variants at two key residues in the coat protein which have been implicated as important to controlling VLP size and geometry. By adapting a method for systematic mutagenesis coupled with size-based selections and high-throughput sequencing as a readout, we developed a quantitative assessment of two residues in MS2 coat protein that govern the size shift in MS2 VLPs. We then applied the strategy to the equivalent residues in Qß VLPs, an MS2 homolog, and demonstrate that the analogous pair of residues are also able to impact Qß VLP size and shape. These results underscore the power of fitness landscapes in identifying critical features for assembly.

Asunto(s)

Vacunas de Partículas Similares a Virus , Tamaño de la Partícula , Biblioteca de Genes , Vacunas de Partículas Similares a Virus/genética

RESUMEN

A critical issue to understanding how populations adapt to new selective pressures is the relative contribution of the initial standing genetic diversity versus that generated de novo. RNA viruses are an excellent model to study this question, as they form highly heterogeneous populations whose genetic diversity can be modulated by factors such as the number of generations, the size of population bottlenecks, or exposure to new environment conditions. In this work, we propagated at nonoptimal temperature (43 °C) two bacteriophage Qß populations differing in their degree of heterogeneity. Deep sequencing analysis showed that, prior to the temperature change, the most heterogeneous population contained some low-frequency mutations that had previously been detected in the consensus sequences of other Qß populations adapted to 43 °C. Evolved populations with origin in this ancestor reached similar growth rates, but the adaptive pathways depended on the frequency of these standing mutations and the transmission bottleneck size. In contrast, the growth rate achieved by populations with origin in the less heterogeneous ancestor did depend on the transmission bottleneck size. The conclusion is that viral diversification in a particular environment may lead to the emergence of mutants capable of accelerating adaptation when the environment changes.

Asunto(s)

Bacteriófagos , Virus ARN , Aclimatación , Adaptación Fisiológica/genética , Bacteriófagos/genética , Variación Genética , Mutación , Virus ARN/genética

RESUMEN

Bovine leukemia virus (BLV) is a C-type retrovirus of cattle that causes huge economic losses with high infection rates in the majority of countries worldwide. To develop an anti-BLV vaccine, we constructed a peptide conjugate using the envelope glycoprotein gp51-peptide epitope, a putative receptor-binding site. This highly antigenic peptide was covalently linked to a mutant bacteriophage carrier (mQß) using two different linker strategies, isothiocyanate (NCS) and dinitrophenyl adipate. Both constructs elicited higher anti-BLV peptide IgG titers than the corresponding conjugate with keyhole limpet hemocyanin protein carrier (gold standard) in mice with the NCS linker strategy requiring less sample processing. The mQß-gp51-peptide construct is the first BLV peptide-based vaccine candidate to generate durable immunity (>539 days), which recognized both native gp51 protein and BLV particles and significantly decreased fusion of a susceptible cell line exposed to infectious BLV. These results support the high translational and animal health potential of the vaccine construct.

Asunto(s)

Virus de la Leucemia Bovina , Animales , Anticuerpos Neutralizantes , Bovinos , Epítopos , Virus de la Leucemia Bovina/metabolismo , Ratones , Péptidos , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

Vibrio cholerae, a noninvasive mucosal pathogen, is endemic in more than 50 countries. Oral cholera vaccines, based on killed whole-cell strains of Vibrio cholerae, can provide significant protection in adults and children for 2-5 years. However, they have relatively limited direct protection in young children. To overcome current challenges, in this study, a potential conjugate vaccine was developed by linking O-specific polysaccharide (OSP) antigen purified from V. cholerae O1 El Tor Inaba strain PIC018 with Qß virus-like particles efficiently via squarate chemistry. The Qß-OSP conjugate was characterized with mass photometry (MP) on the whole particle level. Pertinent immunologic display of OSP was confirmed by immunoreactivity of the conjugate with convalescent phase samples from humans with cholera. Mouse immunization with the Qß-OSP conjugate showed that the construct generated prominent and long-lasting IgG antibody responses against OSP, and the resulting antibodies could recognize the native lipopolysaccharide from Vibrio cholerae O1 Inaba. This was the first time that Qß was conjugated with a bacterial polysaccharide for vaccine development, broadening the scope of this powerful carrier.

Asunto(s)

Vacunas contra el Cólera , Cólera , Vibrio cholerae O1 , Animales , Anticuerpos Antibacterianos , Cólera/microbiología , Cólera/prevención & control , Vacunas contra el Cólera/química , Inmunoglobulina A , Inmunoglobulina G , Inmunoglobulina M , Ratones , Antígenos O

RESUMEN

The broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qß conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qß), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qß-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qß conjugates synthesized represent a promising candidate of HIV-1 vaccine.

Asunto(s)

Allolevivirus/inmunología , Glicopéptidos/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Antígenos/inmunología , Epítopos/genética , Epítopos/inmunología , Glicopéptidos/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fagocitosis/inmunología

RESUMEN

The quadrilateral space is a confined area through which the axillary nerve and posterior circumflex humeral artery (PCHA) travel in the shoulder. Both structures are susceptible to impingement and compression as they travel though this space resulting in a constellation of symptoms known as quadrilateral space syndrome (QSS). Patients may experience paresthesias, loss of motor function, pain and vascular complications. Individuals who perform repetitive overhead arm movements such as elite athletes are at greater risk of developing QSS. The diagnosis can be difficult, but in the setting of clinical suspicion, physical exam and imaging studies can provide specific findings. On MRI, patients may have atrophy of the deltoid or teres minor muscles and angiography may show aneurysm or vascular occlusion of the PCHA. Treatment is initially conservative, with physiotherapy. Surgical decompression is effective in patients with severe or progressive symptoms. Causes of external compression such as fibrous bands, scarring, or other space occupying lesion may be addressed at that time. Neurolysis and aneurysm resection may also be performed. In some cases, emboli from the PCHA can cause ischemia in the involved upper extremity resulting in an acute presentation. Catheter directed therapy such as thrombolysis or thrombectomy may performed emergently in these cases. Though rare, in patients presenting with arm weakness, paresthesia, pain and/or arterial thrombosis in the arm, QSS is an important entity to consider.

RESUMEN

Mass production of transition-metal dichalcogenides has attracted much attention to replace platinum-based catalysts for the hydrogen evolution reaction (HER). Herein, we demonstrate a general strategy for the scalable production of the intrinsic tungsten dichalcogenide (WX2(X = S, Se, Te)) quantum sheets (QSs) by an all-physical top-down method. The method combines silica-assisted ball-milling and sonication-assisted solvent exfoliation and thus enables production of WS2QSs, WSe2QSs, and WTe2QSs in exceedingly high yields of 28.2, 21.3, 19.9 wt%, respectively. The WX2QSs are confirmed as intrinsic and defect-free, which could be determinative to their improved HER performance. The overpotentials of 285, 331, 435 mV at the current density of 10 mA cm-2and Tafel slopes of 116, 78, 162 mV dec-1in acidic media, as well as charge transfer resistance values of 171, 242, 1973 Ω, are derived for WS2QSs, WSe2QSs, and WTe2QSs, respectively, which are much better than those of bulk materials. The WX2QSs exhibit high stability during the electrocatalysis as well. This work offers a powerful approach for fabrication of intrinsic QSs as efficient and robust electrocatalysts.

RESUMEN

Evolution of RNA bacteriophages of the family Leviviridae is governed by the high error rates of their RNA-dependent RNA polymerases. This fact, together with their large population sizes, leads to the generation of highly heterogeneous populations that adapt rapidly to most changes in the environment. Throughout adaptation, the different mutants that make up a viral population compete with each other in a non-trivial process in which their selective values change over time due to the generation of new mutations. In this work we have characterised the intra-population dynamics of a well-studied levivirus, Qß, when it is propagated at a higher-than-optimal temperature. Our results show that adapting populations experienced rapid changes that involved the ascent of particular genotypes and the loss of some beneficial mutations of early generation. Artificially reconstructed populations, containing a fraction of the diversity present in actual populations, fixed mutations more rapidly, illustrating how population bottlenecks may guide the adaptive pathways. The conclusion is that, when the availability of beneficial mutations under a particular selective condition is elevated, the final outcome of adaptation depends more on the occasional occurrence of population bottlenecks and how mutations combine in genomes than on the selective value of particular mutations.

Asunto(s)

Adaptación Biológica , Fagos ARN/fisiología , Temperatura , Evolución Biológica , Evolución Molecular , Regulación Viral de la Expresión Génica , Genoma Viral , Genómica/métodos , Mutación , ARN Viral/genética , Selección Genética

RESUMEN

BACKGROUND: Virus-like Particles (VLPs) are non-genetic multimeric nanoparticles synthesized through in vitro or in vivo self-assembly of one or more viral structural proteins. Immunogenicity and safety of VLPs make them ideal candidates for vaccine development and efficient nanocarriers for foreign antigens or adjuvants to activate the immune system. AIMS: The present study aimed to design and synthesize a chimeric VLP vaccine of the phage Qbeta (Qß) coat protein presenting the universal epitope of the coronavirus. METHODS: The RNA phage Qß coat protein was designed and synthesized, denoted as Qbeta. The CoV epitope, a universal epitope of coronavirus, was inserted into the C-terminal of Qbeta using genetic recombination, designated as Qbeta-CoV. The N-terminal of Qbeta-CoV was successively inserted into the TEV restriction site using mCherry red fluorescent label and modified affinity purified histidine label 6xHE, which was denoted as HE-Qbeta-CoV. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) assessment revealed the expression of Qbeta, Qbeta-CoV, and HE-Qbeta-CoV in the BL21 (DE3) cells. The fusion protein was purified by salting out using ammonium sulfate and affinity chromatography. The morphology of particles was observed using electron microscopy. The female BALB/C mice were immunized intraperitoneally with the Qbeta-CoV and HE-Qbeta-- CoV chimeric VLPs vaccines and their sera were collected for the detection of antibody level and antibody titer using ELISA. The serum is used for the neutralization test of the three viruses of MHV, PEDV, and PDCoV. RESULTS: The results revealed that the fusion proteins Qbeta, Qbeta-CoV, and HE-Qbeta-CoV could all obtain successful expression. Particles with high purity were obtained after purification; the chimeric particles of Qbeta-CoV and HE-Qbeta-CoV were found to be similar to Qbeta particles in morphology and formed chimeric VLPs. In addition, two chimeric VLP vaccines induced specific antibody responses in mice and the antibodies showed certain neutralizing activity. CONCLUSION: The successful construction of the chimeric VLPs of the phage Qß coat protein presenting the universal epitope of coronavirus provides a vaccine form with potential clinical applications for the treatment of coronavirus disease.

Asunto(s)

Anticuerpos Neutralizantes/inmunología , Proteínas de la Cápside/inmunología , Coronavirus/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Filogenia , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Vacunas de Partículas Similares a Virus/genética , Proteínas Virales/genética

RESUMEN

Seed size and shape traits are important determinants of seed yield and appearance quality in soybean [Glycine max (L.) Merr.]. Understanding the genetic architecture of these traits is important to enable their genetic improvement through efficient and targeted selection in soybean breeding, and for the identification of underlying causal genes. To map seed size and shape traits in soybean, a recombinant inbred line (RIL) population developed from K099 (small seed size) × Fendou 16 (large seed size), was phenotyped in three growing seasons. A genetic map of the RIL population was developed using 1,485 genotyping by random amplicon sequencing-direct (GRAS-Di) and 177 SSR markers. Quantitative trait locus (QTL) mapping was conducted by inclusive composite interval mapping. As a result, 53 significant QTLs for seed size traits and 27 significant QTLs for seed shape traits were identified. Six of these QTLs (qSW8.1, qSW16.1, qSLW2.1, qSLT2.1, qSWT1.2, and qSWT4.3) were identified with LOD scores of 3.80-14.0 and R 2 of 2.36%-39.49% in at least two growing seasons. Among the above significant QTLs, 24 QTLs were grouped into 11 QTL clusters, such as, three major QTLs (qSL2.3, qSLW2.1, and qSLT2.1) were clustered into a major QTL on Chr.02, named as qSS2. The effect of qSS2 was validated in a pair of near isogenic lines, and its candidate genes (Glyma.02G269400, Glyma.02G272100, Glyma.02G274900, Glyma.02G277200, and Glyma.02G277600) were mined. The results of this study will assist in the breeding programs aiming at improvement of seed size and shape traits in soybean.

RESUMEN

Phage display technology involves the surface genetic engineering of phages to expose desirable proteins or peptides whose gene sequences are packaged within phage genomes, thereby rendering direct linkage between genotype with phenotype feasible. This has resulted in phage display systems becoming invaluable components of directed evolutionary biotechnology. The M13 is a DNA phage display system which dominates this technology and usually involves selected proteins or peptides being displayed through surface engineering of its minor coat proteins. The displayed protein or peptide's functionality is often highly reduced due to harsh treatment of M13 variants. Recently, we developed a novel phage display system using the coliphage Qß as a nano-biotechnology platform. The coliphage Qß is an RNA phage belonging to the family of Leviviridae, a long investigated virus. Qß phages exist as a quasispecies and possess features making them comparatively more suitable and unique for directed evolutionary biotechnology. As a quasispecies, Qß benefits from the promiscuity of its RNA dependent RNA polymerase replicase, which lacks proofreading activity, and thereby permits rapid variant generation, mutation, and adaptation. The minor coat protein of Qß is the readthrough protein, A1. It shares the same initiation codon with the major coat protein and is produced each time the ribosome translates the UGA stop codon of the major coat protein with the of misincorporation of tryptophan. This misincorporation occurs at a low level (1/15). Per convention and definition, A1 is the target for display technology, as this minor coat protein does not play a role in initiating the life cycle of Qß phage like the pIII of M13. The maturation protein A2 of Qß initiates the life cycle by binding to the pilus of the F+ host bacteria. The extension of the A1 protein with a foreign peptide probe recognizes and binds to the target freely, while the A2 initiates the infection. This avoids any disturbance of the complex and the necessity for acidic elution and neutralization prior to infection. The combined use of both the A1 and A2 proteins of Qß in this display system allows for novel bio-panning, in vitro maturation, and evolution. Additionally, methods for large library size construction have been improved with our directed evolutionary phage display system. This novel phage display technology allows 12 copies of a specific desired peptide to be displayed on the exterior surface of Qß in uniform distribution at the corners of the phage icosahedron. Through the recently optimized subtractive bio-panning strategy, fusion probes containing up to 80 amino acids altogether with linkers, can be displayed for target selection. Thus, combined uniqueness of its genome, structure, and proteins make the Qß phage a desirable suitable innovation applicable in affinity maturation and directed evolutionary biotechnology. The evolutionary adaptability of the Qß phage display strategy is still in its infancy. However, it has the potential to evolve functional domains of the desirable proteins, glycoproteins, and lipoproteins, rendering them superior to their natural counterparts.

Asunto(s)

Biotecnología/métodos , Colifagos/genética , Evolución Molecular Dirigida/métodos , ARN Viral/genética , Técnicas de Visualización de Superficie Celular , Cuasiespecies

RESUMEN

Human papillomavirus (HPV) is a globally prevalent sexually-transmitted pathogen, responsible for most cases of cervical cancer. HPV vaccination rates remain suboptimal, partly due to the need for multiple doses, leading to a lack of compliance and incomplete protection. To address the drawbacks of current HPV vaccines, we used a scalable manufacturing process to prepare implantable polymer-protein blends for single-administration with sustained delivery. Peptide epitopes from HPV16 capsid protein L2 were conjugated to the virus-like particles derived from bacteriophage Qß, to enhance their immunogenicity. The HPV-Qß particles were then encapsulated into poly(lactic-co-glycolic acid) (PLGA) implants, using a benchtop melt-processing system. The implants facilitated the slow and sustained release of HPV-Qß particles without the loss of nanoparticle integrity, during high temperature melt processing. Mice vaccinated with the implants generated IgG titers comparable to the traditional soluble injections and achieved protection in a pseudovirus neutralization assay. HPV-Qß implants offer a new vaccination platform; because the melt-processing is so versatile, the technology offers the opportunity for massive upscale into any geometric form factor. Notably, microneedle patches would allow for self-administration in the absence of a healthcare professional, within the developing world. The Qß technology is highly adaptable, allowing the production of vaccine candidates and their delivery devices for multiple strains or types of viruses.