RESUMEN
The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/clasificación , Linfocitos B/citología , Linfocitos B/metabolismo , Cristalografía por Rayos X , Femenino , Células HEK293 , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/clasificación , VIH-1/metabolismo , Humanos , Macaca mulatta , Masculino , Péptidos/química , Estructura Terciaria de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti-HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Mutación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/genética , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos Anti-VIH/genética , VIH-1/genética , Humanos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that causes severe and often lethal respiratory illness in humans. The MERS-CoV spike (S) protein is the viral fusogen and the target of neutralizing antibodies, and has therefore been the focus of vaccine design efforts. Currently there are no licensed vaccines against MERS-CoV and only a few candidates have advanced to Phase I clinical trials. Here we developed MERS-CoV vaccines utilizing a computationally designed protein nanoparticle platform that has generated safe and immunogenic vaccines against various enveloped viruses, including a licensed vaccine for SARS-CoV-2. Two-component protein nanoparticles displaying MERS-CoV S-derived antigens induced robust neutralizing antibody responses and protected mice against challenge with mouse-adapted MERS-CoV. Electron microscopy polyclonal epitope mapping and serum competition assays revealed the specificities of the dominant antibody responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle vaccine elicited antibodies targeting multiple non-overlapping epitopes in the RBD, whereas anti-NTD antibodies elicited by the S-2P- and NTD-based immunogens converged on a single antigenic site. Our findings demonstrate the potential of two-component nanoparticle vaccine candidates for MERS-CoV and suggest that this platform technology could be broadly applicable to betacoronavirus vaccine development.
RESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a plasma protein that controls cholesterol homeostasis. Here, we design a human PCSK9 mimic, named HIT01, with no consecutive 9-residue stretch in common with any human protein as a potential heart attack vaccine. Murine immunizations with HIT01 reduce low-density lipoprotein (LDL) and cholesterol levels by 40% and 30%, respectively. Immunization of cynomolgus macaques with HIT01-K21Q-R218E, a cleavage-resistant variant, elicits high-titer PCSK9-directed antibody responses and significantly reduces serum levels of cholesterol 2 weeks after each immunization. However, HIT01-K21Q-R218E immunizations also increase serum PCSK9 levels by up to 5-fold, likely due to PCSK9-binding antibodies altering the half-life of PCSK9. While vaccination with a PCSK9 mimic can induce antibodies that block interactions of PCSK9 with the LDL receptor, PCSK9-binding antibodies appear to alter homeostatic levels of PCSK9, thereby confounding its vaccine impact. Our results nevertheless suggest a mechanism for increasing the half-life of soluble regulatory factors by vaccination.
Asunto(s)
Colesterol , Inmunización , Macaca fascicularis , Proproteína Convertasa 9 , Proproteína Convertasa 9/inmunología , Proproteína Convertasa 9/metabolismo , Animales , Humanos , Ratones , Colesterol/metabolismo , Colesterol/sangre , Inmunización/métodos , Receptores de LDL/metabolismo , Femenino , Ratones Endogámicos C57BLRESUMEN
Both protein nanoparticle and mRNA vaccines were clinically de-risked during the COVID-19 pandemic1-6. These vaccine modalities have complementary strengths: antigen display on protein nanoparticles can enhance the magnitude, quality, and durability of antibody responses7-10, while mRNA vaccines can be rapidly manufactured11 and elicit antigen-specific CD4 and CD8 T cells12,13. Here we leverage a computationally designed icosahedral protein nanoparticle that was redesigned for optimal secretion from eukaryotic cells14 to develop an mRNA-launched nanoparticle vaccine for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD)15, formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies.
RESUMEN
Antibody CAP256-VRC26.25 targets the second hypervariable region (V2) at the apex of the HIV envelope (Env) trimer with extraordinary neutralization potency, although less than optimal breadth. To improve breadth, we linked the light chain of CAP256V2LS, an optimized version of CAP256-VRC26.25 currently under clinical evaluation, to the llama nanobody J3, which has broad CD4-binding site-directed neutralization. The J3-linked bispecific antibody exhibited improved breadth and potency over both J3 and CAP256V2LS, indicative of synergistic neutralization. The cryo-EM structure of the bispecific antibody in complex with a prefusion-closed Env trimer revealed simultaneous binding of J3 and CAP256V2LS. We further optimized the pharmacokinetics of the bispecific antibody by reducing the net positive charge of J3. The optimized bispecific antibody, which we named CAP256.J3LS, had a half-life similar to CAP256V2LS in human FcRn knock-in mice and exhibited suitable auto-reactivity, manufacturability, and biophysical risk. CAP256.J3LS neutralized over 97% of a multiclade 208-strain panel (geometric mean concentration for 80% inhibition (IC80) 0.079 µg/ml) and 100% of a 100-virus clade C panel (geometric mean IC80 of 0.05 µg/ml), suggesting its anti-HIV utility especially in regions where clade C dominates.
Asunto(s)
Anticuerpos Biespecíficos , Infecciones por VIH , VIH-1 , Humanos , Animales , Ratones , Anticuerpos Neutralizantes , Pruebas de Neutralización , Anticuerpos Anti-VIH , Sitios de UniónRESUMEN
The broadly neutralizing antibody (bNAb) CAP256-VRC26.25 has exceptional potency against HIV-1 and has been considered for clinical use. During the characterization and production of this bNAb, we observed several unusual features. First, the antibody appeared to adhere to pipette tips, requiring tips to be changed during serial dilution to accurately measure potency. Second, during production scale-up, proteolytic cleavage was discovered to target an extended heavy chain loop, which was attributed to a protease in spent medium from 2-week culture. To enable large scale production, we altered the site of cleavage via a single amino acid change, K100mA. The resultant antibody retained potency and breadth while avoiding protease cleavage. We also added the half-life extending mutation LS, which improved the in vivo persistence in animal models, but did not impact neutralization activity; we observed the same preservation of neutralization for bNAbs VRC01, N6, and PGDM1400 with LS on a 208-virus panel. The final engineered antibody, CAP256V2LS, retained the extraordinary neutralization potency of the parental antibody, had a favorable pharmacokinetic profile in animal models, and was negative in in vitro assessment of autoreactivity. CAP256V2LS has the requisite potency, developability and suitability for scale-up, allowing its advancement as a clinical candidate.
Asunto(s)
Infecciones por VIH , VIH-1 , Animales , Anticuerpos ampliamente neutralizantes , Semivida , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Péptido Hidrolasas , AminoácidosRESUMEN
Antibody-Framework-to-Antigen Distance (AFAD) - the distance between the body of an antibody and a protein antigen - is an important parameter governing antibody recognition. Here, we quantify AFAD for ~2,000 non-redundant antibody-protein-antigen complexes in the Protein Data Bank. AFADs showed a gaussian distribution with mean of 16.3 Å and standard deviation (σ) of 2.4 Å. Notably, antibody-antigen complexes with extended AFADs (>3σ) were exclusively human immunodeficiency virus-type 1 (HIV-1)-neutralizing antibodies. High correlation (R2 = 0.8110) was observed between AFADs and glycan coverage, as assessed by molecular dynamics simulations of the HIV-1-envelope trimer. Especially long AFADs were observed for antibodies targeting the glycosylated trimer apex, and we tested the impact of introducing an apex-glycan hole (N160K); the cryo-EM structure of the glycan hole-targeting HIV-1-neutralizing antibody 2909 in complex with an N160K-envelope trimer revealed a substantially shorter AFAD. Overall, extended AFADs exclusively recognized densely glycosylated surfaces, with the introduction of a glycan hole enabling closer recognition.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Simulación de Dinámica MolecularRESUMEN
Neutralizing antibodies elicited by HIV-1 coevolve with viral envelope proteins (Env) in distinctive patterns, in some cases acquiring substantial breadth. We report that primary HIV-1 envelope proteins-when expressed by simian-human immunodeficiency viruses in rhesus macaques-elicited patterns of Env-antibody coevolution very similar to those in humans, including conserved immunogenetic, structural, and chemical solutions to epitope recognition and precise Env-amino acid substitutions, insertions, and deletions leading to virus persistence. The structure of one rhesus antibody, capable of neutralizing 49% of a 208-strain panel, revealed a V2 apex mode of recognition like that of human broadly neutralizing antibodies (bNAbs) PGT145 and PCT64-35S. Another rhesus antibody bound the CD4 binding site by CD4 mimicry, mirroring human bNAbs 8ANC131, CH235, and VRC01. Virus-antibody coevolution in macaques can thus recapitulate developmental features of human bNAbs, thereby guiding HIV-1 immunogen design.
Asunto(s)
Coevolución Biológica/inmunología , Anticuerpos ampliamente neutralizantes , Anticuerpos Anti-VIH , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Sitios de Unión , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos ampliamente neutralizantes/inmunología , Antígenos CD4/inmunología , Microscopía por Crioelectrón , Epítopos/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Macaca mulatta , Imitación Molecular/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Replicación ViralRESUMEN
The HIV-1 envelope glycoprotein (Env) trimer, composed of gp120 and gp41 subunits, mediates viral entry into cells. Recombinant Env trimers have been studied structurally, but characterization of Env embedded in intact virus membranes has been limited to low resolution. Here, we deploy cryo-electron tomography and subtomogram averaging to determine the structures of Env trimers on aldrithiol-2 (AT-2)-inactivated virions in ligand-free, antibody-bound and CD4-bound forms at subnanometer resolution. Tomographic reconstructions document molecular features consistent with high-resolution structures of engineered soluble and detergent-solubilized Env trimers. One of three conformational states previously predicted by smFRET was not observed by cryo-ET, potentially owing to AT-2 inactivation. We did observe Env trimers to open in situ in response to CD4 binding, with an outward movement of gp120-variable loops and an extension of a critical gp41 helix. Overall features of Env trimer embedded in AT-2-treated virions appear well-represented by current engineered trimers.
Asunto(s)
2,2'-Dipiridil/análogos & derivados , Disulfuros/farmacología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , VIH-1/efectos de los fármacos , Virión/efectos de los fármacos , 2,2'-Dipiridil/farmacología , Línea Celular , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Proteína gp120 de Envoltorio del VIH/ultraestructura , Proteína gp41 de Envoltorio del VIH/ultraestructura , Infecciones por VIH/virología , VIH-1/química , Humanos , Modelos Moleculares , Oxidantes/farmacología , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Solubilidad , Virión/químicaRESUMEN
Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer-stabilized in the prefusion conformation and fused with SpyCatcher-could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with 0.08 µg of SARS-CoV-2 spike-LuS nanoparticle elicited similar neutralizing responses as 2.0 µg of spike, which was ~ 25-fold higher on a weight-per-weight basis. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.
Asunto(s)
Antígenos/inmunología , Betacoronavirus/metabolismo , Nanopartículas/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Antígenos/genética , Antígenos/metabolismo , Aquifex , Bacterias/enzimología , Proteínas Bacterianas/genética , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus , Ferritinas/genética , Helicobacter pylori/metabolismo , Humanos , Ratones , Complejos Multienzimáticos/genética , Pruebas de Neutralización , Pandemias , Neumonía Viral , Multimerización de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Propiedades de SuperficieRESUMEN
Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer - stabilized in the prefusion conformation and fused with SpyCatcher - could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with the SARS-CoV-2 spike-LuS nanoparticles elicited ~25-fold higher neutralizing responses, weight-per-weight relative to spike alone. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.
RESUMEN
The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) - when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) - to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.
Asunto(s)
Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Adyuvantes Inmunológicos , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas/inmunología , Femenino , Inmunización , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Neutralización , Péptidos/químicaRESUMEN
Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.
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Linfocitos B , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos ampliamente neutralizantes/inmunología , Cristalografía por Rayos X , Expresión Génica , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Infecciones por VIH/inmunología , Humanos , Mutación , Transcriptoma/genética , Proteínas Virales de Fusión/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Adipose derived stem cells (ASCs) can be obtained from lipoaspirates and induced in vitro to differentiate into bone, cartilage, and fat. Using this powerful model system we show that after in vitro adipose differentiation a population of cells retain stem-like qualities including multipotency. They are lipid (-), retain the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts. However, these cells are not traditional stem cells because gene expression analysis showed an overall expression profile similar to that of adipocytes. In addition to broadening our understanding of cellular multipotency, our work may be particularly relevant to obesity-associated metabolic disorders. The adipose expandability hypothesis proposes that inability to differentiate new adipocytes is a primary cause of metabolic syndrome in obesity, including diabetes and cardiovascular disease. Here we have defined a differentiation-resistant stem-like multipotent cell population that may be involved in regulation of adipose expandability in vivo and may therefore play key roles in the comorbidities of obesity.