RESUMEN
The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.
Asunto(s)
Vacunas contra el SIDA , VIH-1 , Animales , Humanos , Anticuerpos ampliamente neutralizantes , Antígenos CD4 , Moléculas de Adhesión Celular , VIH-1/fisiología , Macaca , Vacunas contra el SIDA/inmunologíaRESUMEN
A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Linfocitos B , Anticuerpos Anti-VIH , VIH-1 , Humanos , Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Linaje de la Célula , Liposomas , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Mutación , Proteína gp41 de Envoltorio del VIH/inmunologíaRESUMEN
Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , VIH-1/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Polisacáridos/inmunología , SARS-CoV-2/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , COVID-19/inmunología , Dimerización , Epítopos/inmunología , Glicosilación , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Macaca mulatta , Polisacáridos/química , Receptores de Antígenos de Linfocitos B/química , Virus de la Inmunodeficiencia de los Simios/genética , Vacunas/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The SARS-CoV-2 pandemic that causes COVID-19 respiratory syndrome has caused global public health and economic crises, necessitating rapid development of vaccines and therapeutic countermeasures. The world-wide response to the COVID-19 pandemic has been unprecedented with government, academic, and private partnerships working together to rapidly develop vaccine and antibody countermeasures. Many of the technologies being used are derived from prior government-academic partnerships for response to other emerging infections.
Asunto(s)
Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/prevención & control , Vacunas Virales/inmunología , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Betacoronavirus/fisiología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/inmunología , Humanos , Colaboración Intersectorial , Neumonía Viral/inmunología , SARS-CoV-2 , Vacunas Virales/química , Tratamiento Farmacológico de COVID-19RESUMEN
A recombinant lineage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, named XBB, appeared in late 2022 and evolved descendants that successively swept local and global populations. XBB lineage members were noted for their improved immune evasion and transmissibility. Here, we determine cryoelectron microscopy (cryo-EM) structures of XBB.1.5, XBB.1.16, EG.5, and EG.5.1 spike (S) ectodomains to reveal reinforced 3-receptor binding domain (RBD)-down receptor-inaccessible closed states mediated by interprotomer RBD interactions previously observed in BA.1 and BA.2. Improved XBB.1.5 and XBB.1.16 RBD stability compensated for stability loss caused by early Omicron mutations, while the F456L substitution reduced EG.5 RBD stability. S1 subunit mutations had long-range impacts on conformation and epitope presentation in the S2 subunit. Our results reveal continued S protein evolution via simultaneous optimization of multiple parameters, including stability, receptor binding, and immune evasion, and the dramatic effects of relatively few residue substitutions in altering the S protein conformational landscape.
Asunto(s)
COVID-19 , Microscopía por Crioelectrón , Mutación , Conformación Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Humanos , COVID-19/virología , COVID-19/inmunología , Unión Proteica , Evasión Inmune , Modelos Moleculares , Dominios Proteicos , Sitios de UniónRESUMEN
HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of â¼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.
Asunto(s)
Anticuerpos Neutralizantes/genética , Linfocitos B/inmunología , Evolución Molecular , Infecciones por VIH/inmunología , VIH-1/inmunología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Diversidad de Anticuerpos , Enfermedad Crónica , Humanos , Leucocitos Mononucleares , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.
Asunto(s)
COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Humanos , Mutación , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/prevención & control , ARN Mensajero/inmunología , ARN Viral/inmunología , Vacunas Virales/administración & dosificación , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/virología , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Modelos Animales de Enfermedad , Furina/genética , Furina/inmunología , Humanos , Inmunidad Humoral/efectos de los fármacos , Inmunización/métodos , Inmunogenicidad Vacunal , Memoria Inmunológica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Nanopartículas/química , Neumonía Viral/inmunología , Neumonía Viral/patología , ARN Mensajero/genética , ARN Viral/genética , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas , Vacunas Virales/biosíntesis , Vacunas Virales/genéticaRESUMEN
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 AminoácidoRESUMEN
Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Betacoronavirus/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Resfriado Común/prevención & control , Reacciones Cruzadas/inmunología , Pandemias , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Administración Intranasal , Animales , COVID-19/epidemiología , Vacunas contra la COVID-19/inmunología , Resfriado Común/inmunología , Resfriado Común/virología , Modelos Animales de Enfermedad , Femenino , Humanos , Macaca/inmunología , Masculino , Modelos Moleculares , Nanopartículas/química , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Tráquea , VacunaciónRESUMEN
Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing, yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of NTD non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate NTD non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb prophylactic infusion did not suppress infectious viral titers in the lung as potently as neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection from SARS-CoV-2 infection. For therapeutic administration of antibodies, non-nAb effector functions contributed to virus suppression and lessening of lung discoloration, but the presence of neutralization was required for optimal protection from disease. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Fragmentos Fc de Inmunoglobulinas , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , SARS-CoV-2/inmunología , Ratones , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Humanos , Femenino , Dominios Proteicos/inmunología , Carga Viral , Pulmón/virología , Pulmón/inmunología , Pulmón/patologíaRESUMEN
Nucleoside-modified mRNA technology has revolutionized vaccine development with the success of mRNA COVID-19 vaccines. We used modified mRNA technology for the design of envelopes (Env) to induce HIV-1 broadly neutralizing antibodies (bnAbs). However, unlike SARS-CoV-2 neutralizing antibodies that are readily made, HIV-1 bnAb induction is disfavored by the immune system because of the rarity of bnAb B cell precursors and the cross-reactivity of bnAbs targeting certain Env epitopes with host molecules, thus requiring optimized immunogen design. The use of protein nanoparticles (NPs) has been reported to enhance B cell germinal center responses to HIV-1 Env. Here, we report our experience with the expression of Env-ferritin NPs compared with membrane-bound Env gp160 when encoded by modified mRNA. We found that well-folded Env-ferritin NPs were a minority of the protein expressed by an mRNA design and were immunogenic at 20 µg but minimally immunogenic in mice at 1 µg dose in vivo and were not expressed well in draining lymph nodes (LNs) following intramuscular immunization. In contrast, mRNA encoding gp160 was more immunogenic than mRNA encoding Env-NP at 1 µg dose and was expressed well in draining LN following intramuscular immunization. Thus, analysis of mRNA expression in vitro and immunogenicity at low doses in vivo are critical for the evaluation of mRNA designs for optimal immunogenicity of HIV-1 immunogens.IMPORTANCEAn effective HIV-1 vaccine that induces protective antibody responses remains elusive. We have used mRNA technology for designs of HIV-1 immunogens in the forms of membrane-bound full-length envelope gp160 and envelope ferritin nanoparticle. Here, we demonstrated in a mouse model that the membrane-bound form induced a better response than envelope ferritin nanoparticle because of higher in vivo protein expression. The significance of our research is in highlighting the importance of analysis of mRNA design expression and low-dose immunogenicity studies for HIV-1 immunogens before moving to vaccine clinical trials.
RESUMEN
A major goal for the development of vaccines against rapidly mutating viruses, such as influenza or HIV, is to elicit antibodies with broad neutralization capacity. However, B cell precursors capable of maturing into broadly neutralizing antibodies (bnAbs) can be rare in the immune repertoire. Due to the stochastic nature of B cell receptor (BCR) rearrangement, a limited number of third heavy chain complementary determining region (CDRH3) sequences are identical between different individuals. Thus, in order to successfully engage broadly neutralizing antibody precursors that rely on their CDRH3 loop for antigen recognition, immunogens must be able to tolerate sequence diversity in the B cell receptor repertoire across an entire vaccinated population. Here, we present a combined experimental and computational approach to identify BCRs in the human repertoire with CDRH3 loops predicted to be engaged by a target immunogen. For a given antibody/antigen pair, deep mutational scanning was first used to measure the effect of CDRH3 loop substitution on binding. BCR sequences, isolated experimentally or generated in silico, were subsequently evaluated to identify CDRH3 loops expected to be bound by the candidate immunogen. We applied this method to characterize two HIV-1 germline-targeting immunogens and found differences in the frequencies with which they are expected to engage target B cells, thus illustrating how this approach can be used to evaluate candidate immunogens towards B cell precursors engagement and to inform immunogen optimization strategies for more effective vaccine design.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Humanos , Anticuerpos Anti-VIH , Anticuerpos Neutralizantes , Linfocitos B , Anticuerpos ampliamente neutralizantes , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
Prophylactic and therapeutic drugs are urgently needed to combat coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Over the past year, SARS-CoV-2 neutralizing antibodies have been developed for preventive or therapeutic uses. While neutralizing antibodies target the spike protein, their neutralization potency and breadth vary according to recognition epitopes. Several potent SARS-CoV-2 antibodies have shown degrees of success in preclinical or clinical trials, and the US Food and Drug Administration has issued emergency use authorization for two neutralizing antibody cocktails.Nevertheless, antibody therapy for SARS-CoV-2 still faces potential challenges, including emerging viral variants of concern that have antibody-escape mutations and the potential for antibody-mediated enhancement of infection or inflammation. This review summarizes representative SARS-CoV-2 neutralizing antibodies that have been reported and discusses prospects and challenges for the development of the next generation of COVID-19 preventive or therapeutic antibodies.
Asunto(s)
COVID-19 , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes , Humanos , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
The envelope glycoprotein (Env) is the main focus of human immunodeficiency virus type 1 (HIV-1) vaccine development due to its critical role in viral entry. Despite advances in protein engineering, many Env proteins remain recalcitrant to recombinant expression due to their inherent metastability, making biochemical and immunological experiments impractical or impossible. Here, we report a novel proline stabilization strategy to facilitate the production of prefusion Env trimers. This approach, termed "2P," works synergistically with previously described SOSIP mutations and dramatically increases the yield of recombinantly expressed Env ectodomains without altering the antigenic or conformational properties of near-native Env. We determined that the 2P mutations function by enhancing the durability of the prefusion conformation and that this stabilization strategy is broadly applicable to evolutionarily and antigenically diverse Env constructs. These findings provide a new Env stabilization platform to facilitate biochemical research and expand the number of Env variants that can be developed as future HIV-1 vaccine candidates. IMPORTANCE Recent estimates have placed the number of new human immunodeficiency virus type 1 (HIV-1) infections at approximately 1.5 million per year, emphasizing the ongoing and urgent need for an effective vaccine. The envelope (Env) glycoprotein is the main focus of HIV-1 vaccine development, but, due to its inherent metastability, many Env variants are difficult to recombinantly express in the relatively large quantities that are required for biochemical studies and animal trials. Here, we describe a novel structure-based stabilization strategy that works synergistically with previously described SOSIP mutations to increase the yield of prefusion HIV-1 Env.
Asunto(s)
Glicoproteínas , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Humanos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Glicoproteínas/genética , Infecciones por VIH , Conformación Molecular , Ingeniería de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/genética , VIH-1/genéticaRESUMEN
A primary goal of HIV-1 vaccine development is the consistent elicitation of protective, neutralizing antibodies. While highly similar neutralizing antibodies (nAbs) have been isolated from multiple HIV-infected individuals, it is unclear whether vaccination can consistently elicit highly similar nAbs in genetically diverse primates. Here, we show in three outbred rhesus macaques that immunization with Env elicits a genotypically and phenotypically conserved nAb response. From these vaccinated macaques, we isolated four antibody lineages that had commonalities in immunoglobulin variable, diversity, and joining gene segment usage. Atomic-level structures of the antigen binding fragments of the two most similar antibodies showed nearly identical paratopes. The Env binding modes of each of the four vaccine-induced nAbs were distinct from previously known monoclonal HIV-1 neutralizing antibodies, but were nearly identical to each other. The similarities of these antibodies show that the immune system in outbred primates can respond to HIV-1 Env vaccination with a similar structural and genotypic solution for recognizing a particular neutralizing epitope. These results support rational vaccine design for HIV-1 that aims to reproducibly elicit, in genetically diverse primates, nAbs with specific paratope structures capable of binding conserved epitopes.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Macaca mulattaRESUMEN
Daily burden and clinical toxicities associated with antiretroviral therapy (ART) emphasize the need for alternative strategies to induce long-term human immunodeficiency virus (HIV) remission upon ART cessation. Broadly neutralizing antibodies (bNAbs) can both neutralize free virions and mediate effector functions against infected cells and therefore represent a leading immunotherapeutic approach. To increase potency and breadth, as well as to limit the development of resistant virus strains, it is likely that bNAbs will need to be administered in combination. It is therefore critical to identify bNAb combinations that can achieve robust polyfunctional antiviral activity against a high number of HIV strains. In this study, we systematically assessed the abilities of single bNAbs and triple bNAb combinations to mediate robust polyfunctional antiviral activity against a large panel of cross-clade simian-human immunodeficiency viruses (SHIVs), which are commonly used as tools for validation of therapeutic strategies targeting the HIV envelope in nonhuman primate models. We demonstrate that most bNAbs are capable of mediating both neutralizing and nonneutralizing effector functions against cross-clade SHIVs, although the susceptibility to V3 glycan-specific bNAbs is highly strain dependent. Moreover, we observe a strong correlation between the neutralization potencies and nonneutralizing effector functions of bNAbs against the transmitted/founder SHIV CH505. Finally, we identify several triple bNAb combinations comprising of CD4 binding site-, V2-glycan-, and gp120-gp41 interface-targeting bNAbs that are capable of mediating synergistic polyfunctional antiviral activities against multiple clade A, B, C, and D SHIVs.IMPORTANCE Optimal bNAb immunotherapeutics will need to mediate multiple antiviral functions against a broad range of HIV strains. Our systematic assessment of triple bNAb combinations against SHIVs will identify bNAbs with synergistic, polyfunctional antiviral activity that will inform the selection of candidate bNAbs for optimal combination designs. The identified combinations can be validated in vivo in future passive immunization studies using the SHIV challenge model.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos ampliamente neutralizantes/uso terapéutico , Anticuerpos Anti-VIH/uso terapéutico , Mutación , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Humanos , Inmunización Pasiva , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 hours following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen density with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in separate nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not observed. Taken together, this study provides a new vaccine platform for administration of glycopeptide immunogens for focusing immune responses to specific bnAb epitopes.
Asunto(s)
Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Nanopartículas/uso terapéutico , Animales , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos/inmunología , Epítopos/inmunología , Femenino , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/inmunología , Seropositividad para VIH/inmunología , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Péptidos , PrimatesRESUMEN
The continuing emergence of viral pathogens and their rapid spread into heavily populated areas around the world underscore the urgency for development of highly effective vaccines to generate protective antiviral Ab responses. Many established and newly emerging viral pathogens, including HIV and Ebola viruses, are most prevalent in regions of the world in which Mycobacterium tuberculosis infection remains endemic and vaccination at birth with M. bovis bacille Calmette-Guérin (BCG) is widely used. We have investigated the potential for using CD4+ T cells arising in response to BCG as a source of help for driving Ab responses against viral vaccines. To test this approach, we designed vaccines comprised of protein immunogens fused to an immunodominant CD4+ T cell epitope of the secreted Ag 85B protein of BCG. Proof-of-concept experiments showed that the presence of BCG-specific Th cells in previously BCG-vaccinated mice had a dose-sparing effect for subsequent vaccination with fusion proteins containing the Ag 85B epitope and consistently induced isotype switching to the IgG2c subclass. Studies using an Ebola virus glycoprotein fused to the Ag 85B epitope showed that prior BCG vaccination promoted high-affinity IgG1 responses that neutralized viral infection. The design of fusion protein vaccines with the ability to recruit BCG-specific CD4+ Th cells may be a useful and broadly applicable approach to generating improved vaccines against a range of established and newly emergent viral pathogens.
Asunto(s)
Aciltransferasas/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/inmunología , Mycobacterium bovis/inmunología , Proteínas del Envoltorio Viral/inmunología , Aciltransferasas/genética , Animales , Anticuerpos Antivirales/metabolismo , Formación de Anticuerpos , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Vacunas contra el Virus del Ébola/genética , Femenino , Humanos , Inmunoglobulina G/sangre , Activación de Linfocitos , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
Small-molecule viral entry inhibitors, such as BMS-626529 (BMS-529), allosterically block CD4 binding to HIV-1 envelope (Env) and inhibit CD4-induced structural changes in Env trimers. Here, we show that the binding of BMS-529 to clade C soluble chimeric gp140 SOSIP (ch.SOSIP) and membrane-bound trimers with intact transmembrane domain (gp150) prevented trimer conformational transitions and enhanced their immunogenicity. When complexed to BMS-529, ch.SOSIP trimers retained their binding to broadly neutralizing antibodies (bNAbs) and to their unmutated common ancestor (UCA) antibodies, while exposure of CD4-induced (CD4i) non-bNAb epitopes was inhibited. BMS-529-complexed gp150 trimers in detergent micelles, which were isolated from CHO cells, bound to bNAbs, including UCA and intermediates of the CD4 binding site (bs) CH103 bNAb lineage, and showed limited exposure of CD4i epitopes and a glycosylation pattern with a preponderance of high-mannose glycans. In rabbits, BMS-529-complexed V3 glycan-targeting ch.SOSIP immunogen induced in the majority of immunized animals higher neutralization titers against both autologous and select high mannose-bearing heterologous tier 2 pseudoviruses than those immunized with the noncomplexed ch.SOSIP. In rhesus macaques, BMS-529 complexed to CD4 bs-targeting ch.SOSIP immunogen induced stronger neutralization against tier 2 pseudoviruses bearing high-mannose glycans than noncomplexed ch.SOSIP trimer immunogen. When immunized with gp150 complexed to BMS-529, rhesus macaques showed neutralization against tier 2 pseudoviruses with targeted glycan deletion and high-mannose glycan enrichment. These results demonstrated that stabilization of Env trimer conformation with BMS-529 improved the immunogenicity of select chimeric SOSIP trimers and elicited tier 2 neutralizing antibodies of higher potency than noncomplexed trimers.IMPORTANCE Soluble forms of HIV-1 envelope trimers exhibit conformational heterogeneity and undergo CD4-induced (CD4i) exposure of epitopes of non-neutralizing antibodies that can potentially hinder induction of broad neutralizing antibody responses. These limitations have been mitigated through recent structure-guided approaches and include trimer-stabilizing mutations that resist trimer conformational transition and exposure of CD4i epitopes. The use of small-molecule viral inhibitors that allosterically block CD4 binding represents an alternative strategy for stabilizing Env trimer in the pre-CD4-triggered state of both soluble and membrane-bound trimers. In this study, we report that the viral entry inhibitor BMS-626529 restricts trimer conformational transition and improves the immunogenicity of select Env trimer immunogens.