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1.
Cell ; 181(2): 281-292.e6, 2020 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-32155444

RESUMEN

The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.


Asunto(s)
Betacoronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Antígenos Virales/química , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Betacoronavirus/química , Línea Celular , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Peptidil-Dipeptidasa A/metabolismo , Receptores Virales/química , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos
2.
Immunity ; 57(6): 1428-1441.e8, 2024 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-38723638

RESUMEN

Induction of commensal-specific immunity contributes to tissue homeostasis, yet the mechanisms underlying induction of commensal-specific B cells remain poorly understood in part due to a lack of tools to identify these cells. Using phage display, we identified segmented filamentous bacteria (SFB) antigens targeted by serum and intestinal antibodies and generated B cell tetramers to track SFB-specific B cells in gut-associated lymphoid tissues. We revealed a compartmentalized response in SFB-specific B cell activation, with a gradient of immunoglobulin A (IgA), IgG1, and IgG2b isotype production along Peyer's patches contrasted by selective production of IgG2b within mesenteric lymph nodes. V(D)J sequencing and monoclonal antibody generation identified somatic hypermutation driven affinity maturation to SFB antigens under homeostatic conditions. Combining phage display and B cell tetramers will enable investigation of the ontogeny and function of commensal-specific B cell responses in tissue immunity, inflammation, and repair.


Asunto(s)
Linfocitos B , Animales , Linfocitos B/inmunología , Ratones , Ratones Endogámicos C57BL , Ganglios Linfáticos Agregados/inmunología , Activación de Linfocitos/inmunología , Antígenos Bacterianos/inmunología , Hipermutación Somática de Inmunoglobulina , Biblioteca de Péptidos , Ganglios Linfáticos/inmunología , Técnicas de Visualización de Superficie Celular , Simbiosis/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina A/inmunología
3.
Cell ; 166(6): 1471-1484.e18, 2016 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-27610571

RESUMEN

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , VIH-1/inmunología , Inmunización , Cadenas Pesadas de Inmunoglobulina/inmunología , Células Precursoras de Linfocitos B/inmunología , Animales , Anticuerpos Monoclonales/genética , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Anticuerpos Anti-VIH , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Concentración 50 Inhibidora , Ratones , Eliminación de Secuencia , Linfocitos T/inmunología
4.
Immunity ; 54(9): 2143-2158.e15, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34453881

RESUMEN

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.


Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Encéfalo/patología , COVID-19/inmunología , Pulmón/patología , SARS-CoV-2/fisiología , Testículo/patología , Enzima Convertidora de Angiotensina 2/genética , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Encéfalo/virología , COVID-19/terapia , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Luciferasas/genética , Mediciones Luminiscentes , Pulmón/virología , Masculino , Ratones , Ratones Transgénicos , Testículo/virología
6.
Cell ; 161(7): 1505-15, 2015 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-26091035

RESUMEN

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.


Asunto(s)
Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Técnicas de Sustitución del Gen , VIH-1/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Animales , Antígenos Virales , Linfocitos B/inmunología , Antígenos CD4/metabolismo , Infecciones por VIH/inmunología , Humanos , Ratones , Mutación , Bazo/citología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
7.
Immunity ; 53(1): 98-105.e5, 2020 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-32561270

RESUMEN

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Hipermutación Somática de Inmunoglobulina/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2 , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Sitios de Unión , COVID-19 , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Epítopos de Linfocito B/inmunología , Humanos , Pandemias/prevención & control , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/inmunología , Neumonía Viral/prevención & control , Unión Proteica , Receptores Virales/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Vacunas Virales/inmunología
8.
Immunity ; 48(4): 799-811.e9, 2018 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-29669253

RESUMEN

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells, the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical, and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas del Envoltorio Viral/inmunología , Células 3T3 , Animales , Linfocitos B/virología , Células CHO , Línea Celular , Cricetulus , Microscopía por Crioelectrón , Células Epiteliales/virología , Epítopos de Linfocito B/inmunología , Células HEK293 , Humanos , Ratones , Acoplamiento Viral
9.
Immunity ; 49(6): 1162-1174.e8, 2018 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-30552024

RESUMEN

Elicitation of VRC01-class broadly neutralizing antibodies (bnAbs) is an appealing approach for a preventative HIV-1 vaccine. Despite extensive investigations, strategies to induce VRC01-class bnAbs and overcome the barrier posed by the envelope N276 glycan have not been successful. Here, we inferred a high-probability unmutated common ancestor (UCA) of the VRC01 lineage and reconstructed the stages of lineage maturation. Env immunogens designed on reverted VRC01-class bnAbs bound to VRC01 UCA with affinity sufficient to activate naive B cells. Early mutations defined maturation pathways toward limited or broad neutralization, suggesting that focusing the immune response is likely required to steer B cell maturation toward the development of neutralization breadth. Finally, VRC01 lineage bnAbs with long CDR H3s overcame the HIV-1 N276 glycan barrier without shortening their CDR L1, revealing a solution for broad neutralization in which the heavy chain, not CDR L1, is the determinant to accommodate the N276 glycan.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Polisacáridos/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/clasificación , Anticuerpos Neutralizantes/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión/genética , Anticuerpos ampliamente neutralizantes , Antígenos CD4/genética , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Anticuerpos Anti-VIH , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Filogenia , Polisacáridos/metabolismo , Homología de Secuencia de Aminoácido
10.
Nature ; 570(7762): 468-473, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31142836

RESUMEN

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.


Asunto(s)
Vacunas contra el SIDA/inmunología , Linfocitos B/inmunología , Células Clonales/inmunología , VIH-1/química , VIH-1/inmunología , Macaca mulatta/inmunología , Vacunación , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/ultraestructura , Afinidad de Anticuerpos , Especificidad de Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Linfocitos B/citología , Proliferación Celular , Células Clonales/citología , Clonación Molecular , Reactividad Cruzada/inmunología , Microscopía por Crioelectrón , Femenino , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/ultraestructura , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/ultraestructura , Activación de Linfocitos , Masculino , Ratones , Modelos Moleculares , Polisacáridos/inmunología , Conejos , Hipermutación Somática de Inmunoglobulina
11.
Immunity ; 43(5): 837-40, 2015 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-26588775

RESUMEN

Broadly neutralizing antibodies targeting quaternary epitopes on the apex of the HIV-1 envelope spike are an attractive vaccine target, yet engineering immunogens that recapitulate such epitopes has proven difficult. In this issue of Immunity, Andrabi and colleagues (2015) identify an exciting new candidate immunogen that could initiate the production of these types of antibodies through vaccination.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Vacunas/inmunología , Proteínas del Envoltorio Viral/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Humanos
12.
J Infect Dis ; 223(11): 1897-1904, 2021 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-33095855

RESUMEN

BACKGROUND: Epstein-Barr virus (EBV) infection is a major cause of malignancy worldwide. Maternal antibody is thought to prevent EBV infection because it is uncommon in early infancy. Maternal HIV infection is associated with an increased incidence of EBV infection in exposed infants, which we hypothesized results from impaired transfer of EBV-neutralizing maternal antibodies. METHODS: Among Ugandan infants followed for EBV acquisition from birth, we measured antibody binding to EBV glycoproteins (gp350, gH/gL) involved in B-cell and epithelial-cell entry, as well as viral neutralization and antibody-dependent cellular cytotoxicity (ADCC) activity in plasma samples prior to infection. These serologic data were analyzed for differences between HIV-exposed uninfected (HEU) and HIV-unexposed (HUU) infants, and for associations with incident infant EBV infection. RESULTS: HEU infants had significantly higher titers than HUU infants for all EBV-binding and neutralizing antibodies measured (P < .01) but not ADCC activity, which was similar between groups. No antibody measure was associated with a decreased risk of EBV acquisition in the cohort. CONCLUSIONS: Our findings indicate that in this cohort maternal antibody did not protect infants against EBV infection through viral neutralization. The identification of protective nonneutralizing antibody functions would be invaluable for the development of an EBV vaccine.


Asunto(s)
Anticuerpos Antivirales/inmunología , Infecciones por Virus de Epstein-Barr , Infecciones por VIH , Inmunidad Materno-Adquirida , Infecciones por Virus de Epstein-Barr/epidemiología , Femenino , Infecciones por VIH/complicaciones , Herpesvirus Humano 4 , Humanos , Lactante , Uganda/epidemiología
14.
Proc Natl Acad Sci U S A ; 115(18): 4743-4748, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29666227

RESUMEN

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , VIH-1/inmunología , Células Precursoras de Linfocitos B/inmunología , Animales , Ratones , Ratones Transgénicos
15.
Immunol Rev ; 275(1): 203-216, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-28133796

RESUMEN

In 2009, Dimitrov's group reported that the inferred germline (iGL) forms of several HIV-1 broadly neutralizing antibodies (bNAbs) did not display measurable binding to a recombinant gp140 Env protein (derived from the dual-tropic 89.6 virus), which was efficiently recognized by the mature (somatically mutated) antibodies. At that time, a small number of bNAbs were available, but in the following years, the implementation of high-throughput B-cell isolation and sequencing assays and of screening methodologies facilitated the isolation of greater numbers of bNAbs from infected subjects. Using these newest bNAbs, and a wide range of diverse recombinant Envs, we and others confirmed the observations made by Dimitrov's group. The results from these studies created a paradigm shift in our collective thinking as to why recombinant Envs are ineffective in eliciting bNAbs and has led to the "germline-targeting" immunization approach. Here we discuss this approach in detail: what has been done so far, the advantages and limitations of the current germline-targeting immunogens and of the animal models used to test them, and we conclude with a few thoughts about future directions in this area of research.


Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Linfocitos B/inmunología , Anticuerpos Anti-VIH/metabolismo , Infecciones por VIH/inmunología , VIH-1/inmunología , Proteínas del Envoltorio Viral/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Linfocitos B/virología , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica , Vacunación
16.
PLoS Pathog ; 14(11): e1007431, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30395637

RESUMEN

Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Linfocitos B/inmunología , Sitios de Unión , Anticuerpos ampliamente neutralizantes , Antígenos CD4/inmunología , Epítopos/inmunología , Glicosilación , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Seropositividad para VIH , VIH-1/inmunología , Humanos , Polisacáridos/inmunología , Polisacáridos/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología
17.
J Virol ; 92(4)2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29093093

RESUMEN

Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5-Å-resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified Deltacoronavirus genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved compared with the human respiratory coronavirus NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2' activation loop, containing a reduced number of basic amino acids, which participates in rendering the spike largely protease resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.IMPORTANCE Coronaviruses use transmembrane S glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic-resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic PDCoV, which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease resistant, which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts.


Asunto(s)
Infecciones por Coronaviridae/virología , Coronaviridae/fisiología , Fusión de Membrana , Polisacáridos/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Acoplamiento Viral , Animales , Coronaviridae/clasificación , Infecciones por Coronaviridae/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/virología , Humanos , Polisacáridos/química , Conformación Proteica , Porcinos , Enfermedades de los Porcinos/patología , Enfermedades de los Porcinos/virología
18.
PLoS Pathog ; 10(5): e1004103, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24788925

RESUMEN

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.


Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Mapeo Epitopo , Hemaglutinación por Virus/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Virus de la Influenza A/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Hemaglutinación por Virus/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Vacunas contra la Influenza/química , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Ingeniería de Proteínas/métodos , Estructura Cuaternaria de Proteína , Homología de Secuencia de Aminoácido
19.
J Virol ; 88(5): 2645-57, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24352455

RESUMEN

UNLABELLED: Broadly neutralizing antibodies (bNAbs) against HIV-1 are generated during HIV-1-infection but have not yet been elicited by immunization with recombinant forms of the viral envelope glycoprotein (Env; the target of anti-HIV-1 neutralizing antibodies). A particular type of bNAb targets the CD4-binding site (CD4-BS) region of Env. These antibodies are derived from a limited number of VH/VL genes and can bind to and neutralize diverse HIV-1 strains. Recent reports have demonstrated the limited potential of Env to activate B cells expressing the germline B cell receptor (BCR) forms of anti-CD4-BS bNAbs. A potential reason for the lack of elicitation of anti-CD4-BS bNAbs by Env immunogens is the absence of stimulation of naive B cells expressing the germline BCRs of such antibodies. Several bNAbs have been isolated from HIV-1-infected subjects that target other structurally conserved regions of Env. How frequently Env immunogens stimulate the germline BCRs that give rise to bNAbs that target Env regions other than the CD4-BS is not well understood. Here, we investigated the interactions between diverse Envs and the BCRs of known bNAbs targeting not only the CD4-BS but also conserved elements of the second and third variable Env regions. Our results indicate that Env is generally ineffective in engaging germline BCRs of bNAbs irrespective of their epitope target. Potentially, this is the result of viral evolutionary mechanisms adopted to escape broadly neutralizing antibody responses. Our results also suggest that a single Env capable of activating germline BCRs that target distinct Env epitopes will be very difficult to identify or to design. IMPORTANCE: Broadly neutralizing antibodies against HIV-1 are thought to be an important component of the immune responses that a successful vaccine should elicit. Broadly neutralizing antibodies are generated by a subset of those infected by HIV-1, but so far, they have not been generated by immunization with recombinant Envelope (Env, the target of anti-HIV-1 neutralizing antibodies). Here, we provide evidence that the inability of Env to elicit the production of broadly neutralizing antibodies is due to the inability of diverse Envs to engage the germline B cell receptor forms of known broadly neutralizing antibodies.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/genética , Linfocitos B/metabolismo , Línea Celular , Eliminación de Gen , Expresión Génica , Variación Genética , Glicosilación , Anticuerpos Anti-VIH/genética , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Mutación , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas Recombinantes/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
20.
PLoS Pathog ; 9(1): e1003106, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23300456

RESUMEN

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Antígenos CD4/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Vacunas contra el SIDA/inmunología , Anticuerpos Antiidiotipos/inmunología , Afinidad de Anticuerpos/inmunología , Antígenos Virales/inmunología , Linfocitos B/inmunología , Línea Celular , Epítopos/inmunología , Células HEK293 , Infecciones por VIH/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Activación de Linfocitos , Pruebas de Neutralización , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología
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