RESUMEN
Antibody complementarity determining region diversity has been considered to be the most important metric for the production of a functional antibody library. Generally, the greater the antibody library diversity, the greater the probability of selecting a diverse array of high affinity leads. According to this paradigm, the primary means of elevating library diversity has been by increasing the number of donors. In the present study we explored the possibility of creating an in vitro antibody library from a single healthy individual, showing that the number of lymphocytes, rather than the number of donors, is the key criterion in the production of a diverse and functional antibody library. We describe the construction of a high-quality phage display library comprising 5 × 109 human antibodies by applying an efficient B cell extraction protocol from a single donor and a targeted V-gene amplification strategy favoring specific antibody families for their improved developability profiles. Each step of the library generation process was followed and validated by next generation sequencing to monitor the library quality and diversity. The functionality of the library was tested using several therapeutically relevant targets for which a vast number of different antibodies with desired biophysical properties were obtained.
Asunto(s)
Anticuerpos/metabolismo , Linfocitos B/metabolismo , Técnicas de Visualización de Superficie Celular , Biblioteca de Péptidos , Anticuerpos/genética , Anticuerpos/inmunología , Especificidad de Anticuerpos , Linfocitos B/inmunología , Voluntarios Sanos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucaféresis , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV and influenza virus, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α-2,3-linked sialic acid and α-2,6-linked sialic acid, respectively. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α-2,3 linkage in H1 subtype viruses; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Ácidos Siálicos/inmunología , Animales , Pollos , Cristalización , Perros , Huevos , Humanos , Inmunogenicidad Vacunal/inmunología , Gripe Aviar/virología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Células Plasmáticas/inmunología , Estructura Terciaria de ProteínaRESUMEN
The influenza C virus CM2 protein and a chimeric influenza A virus M2 protein (MCM) containing the CM2 transmembrane domain were assessed for their ability to functionally replace the M2 protein. While all three proteins could alter cytosolic pH to various degrees when expressed from cDNA, only M2 and MCM could at least partially restore infectious virus production to M2-deficient influenza A viruses. The data suggest that while the CM2 ion channel activity is similar to that of M2, sequences in the extracellular and/or cytoplasmic domains play important roles in infectious virus production.
Asunto(s)
Citoplasma/química , Gammainfluenzavirus/fisiología , Virus de la Influenza A/fisiología , Gripe Humana/virología , Proteínas de la Matriz Viral/metabolismo , Replicación Viral , Animales , Línea Celular , Citoplasma/virología , Humanos , Concentración de Iones de Hidrógeno , Virus de la Influenza A/genética , Gammainfluenzavirus/química , Gammainfluenzavirus/genética , Estructura Terciaria de Proteína , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
Influenza A virus encodes M2, a proton channel that has been shown to be important during virus entry and assembly. In order to systematically investigate the role of the membrane-proximal residues in the M2 cytoplasmic tail in virus replication, we utilized scanning and directed alanine mutagenesis in combination with transcomplementation assays and recombinant viruses. The membrane-proximal residues 46 to 69 tolerated numerous mutations, with little, if any, effect on virus replication, suggesting that protein structure rather than individual amino acid identity in this region may be critical for M2 protein function.
Asunto(s)
Membrana Celular/metabolismo , Citoplasma/metabolismo , Subtipo H3N2 del Virus de la Influenza A/genética , Mutación/genética , Proteínas de la Matriz Viral/genética , Replicación Viral , Secuencia de Aminoácidos , Animales , Western Blotting , Células Cultivadas , Perros , Riñón/citología , Riñón/metabolismo , Riñón/virología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Plásmidos , Homología de Secuencia de Aminoácido , Proteínas de la Matriz Viral/metabolismo , Ensamble de VirusRESUMEN
Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.
Asunto(s)
Epítopos/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Gripe Aviar/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Aves , Reacciones Cruzadas/genética , Reacciones Cruzadas/inmunología , Brotes de Enfermedades , Epítopos/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Aviar/genética , Gripe Humana/genética , Gripe Humana/inmunología , Gripe Humana/prevención & control , Ratones , Datos de Secuencia MolecularRESUMEN
Influenza A virus particles assemble and bud from plasma membrane domains enriched with the viral glycoproteins but only a small fraction of the total M2 protein is incorporated into virus particles when compared to the other viral glycoproteins. A membrane proximal cholesterol recognition/interaction amino acid consensus (CRAC) motif was previously identified in M2 and suggested to play a role in protein function. We investigated the importance of the CRAC motif on virus replication by generating recombinant proteins and viruses containing amino acid substitutions in this motif. Alteration or completion of the M2 CRAC motif in two different virus strains caused no changes in virus replication in vitro. Viruses lacking an M2 CRAC motif had decreased morbidity and mortality in the mouse model of infection, suggesting that this motif is a virulence determinant which may facilitate virus replication in vivo but is not required for basic virus replication in tissue culture.
Asunto(s)
Colesterol/metabolismo , Secuencia de Consenso , Virus de la Influenza A/patogenicidad , Proteínas de la Matriz Viral/química , Replicación Viral , Secuencias de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Células Epiteliales/virología , Femenino , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/virología , Tráquea/citología , Tráquea/virología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Virulencia , Ensamble de VirusRESUMEN
The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.
Asunto(s)
Virus de la Influenza A/fisiología , Tirosina/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Virión/fisiología , Replicación Viral , Secuencias de Aminoácidos , Animales , Línea Celular , Cricetinae , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/genética , Gripe Humana/virología , Mutación , Tirosina/química , Tirosina/genética , Proteínas de la Matriz Viral/genética , Virión/química , Virión/genética , Ensamble de VirusRESUMEN
Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease in humans with high rates of mortality. The virus has a tripartite genome composed of a small (S), a medium (M) and a large (L) RNA segment; the M segment encodes the two viral glycoproteins, G(N) and G(C). Whilst relatively few full-length M segment sequences are available, it is apparent that both G(N) and G(C) may exhibit significant sequence diversity. It is unknown whether considerable antigenic differences exist between divergent CCHFV strains, or whether there are conserved neutralizing epitopes. The M segments derived from viral isolates of a human case of CCHF in South Africa (SPU 41/84), an infected tick (Hyalomma marginatum) in South Africa (SPU 128/81), a human case in Congo (UG 3010), an infected individual in Uzbekistan (U2-2-002) and an infected tick (Hyalomma asiaticum) in China (Hy13) were sequenced fully, and the glycoproteins were expressed. These novel sequences showed high variability in the N-terminal region of G(N) and more modest differences in the remainder of G(N) and in G(C). Phylogenetic analyses placed these newly identified strains in three of the four previously described M segment groups. Studies with a panel of mAbs specific to G(N) and G(C) indicated that there were significant antigenic differences between the M segment groups, although several neutralizing epitopes in both G(N) and G(C) were conserved among all strains examined. Thus, the genetic diversity exhibited by CCHFV strains results in significant antigenic differences that will need to be taken into consideration for vaccine development.