RESUMO
Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Anticorpos Amplamente Neutralizantes , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vacinas Sintéticas/imunologia , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/genética , Antígenos Virais/imunologia , Feminino , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Alinhamento de Sequência , Vacinas Sintéticas/administração & dosagemRESUMO
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.
Assuntos
Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Técnicas de Introdução de Genes , HIV-1/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Antígenos Virais , Linfócitos B/imunologia , Antígenos CD4/metabolismo , Infecções por HIV/imunologia , Humanos , Camundongos , Mutação , Baço/citologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.
Assuntos
COVID-19 , Anticorpos de Domínio Único , Humanos , Anticorpos de Domínio Único/química , Saccharomyces cerevisiae/metabolismo , SARS-CoV-2 , Anticorpos , EpitoposRESUMO
The dense patch of high-mannose-type glycans surrounding the N332 glycan on the HIV envelope glycoprotein (Env) is targeted by multiple broadly neutralizing antibodies (bnAbs). This region is relatively conserved, implying functional importance, the origins of which are not well understood. Here we describe the isolation of new bnAbs targeting this region. Examination of these and previously described antibodies to Env revealed that four different bnAb families targeted the (324)GDIR(327) peptide stretch at the base of the gp120 V3 loop and its nearby glycans. We found that this peptide stretch constitutes part of the CCR5 co-receptor binding site, with the high-mannose patch glycans serving to camouflage it from most antibodies. GDIR-glycan bnAbs, in contrast, bound both (324)GDIR(327) peptide residues and high-mannose patch glycans, which enabled broad reactivity against diverse HIV isolates. Thus, as for the CD4 binding site, bnAb effectiveness relies on circumventing the defenses of a critical functional region on Env.
Assuntos
Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Polissacarídeos/metabolismo , Motivos de Aminoácidos , Antígenos CD4/metabolismo , Mapeamento de Epitopos , Epitopos/metabolismo , Engenharia Genética , Células HEK293 , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Imunidade Humoral , Memória Imunológica , Fragmentos de Peptídeos/metabolismo , Polissacarídeos/imunologia , Ligação Proteica , Receptores CCR5/metabolismoRESUMO
Tools to monitor SARS-CoV-2 transmission and immune responses are needed. We present a neutralization ELISA to determine the levels of Ab-mediated virus neutralization and a preclinical model of focused immunization strategy. The ELISA is strongly correlated with the elaborate plaque reduction neutralization test (ρ = 0.9231, p < 0.0001). The neutralization potency of convalescent sera strongly correlates to IgG titers against SARS-CoV-2 receptor-binding domain (RBD) and spike (ρ = 0.8291 and 0.8297, respectively; p < 0.0001) and to a lesser extent with the IgG titers against protein N (ρ = 0.6471, p < 0.0001). The preclinical vaccine NMRI mice models using RBD and full-length spike Ag as immunogens show a profound Ab neutralization capacity (IC50 = 1.9 × 104 to 2.6 × 104 and 3.9 × 103 to 5.2 × 103, respectively). Using a panel of novel high-affinity murine mAbs, we also show that a majority of the RBD-raised mAbs have inhibitory properties, whereas only a few of the spike-raised mAbs do. The ELISA-based viral neutralization test offers a time- and cost-effective alternative to the plaque reduction neutralization test. The immunization results indicate that vaccine strategies focused only on the RBD region may have advantages compared with the full spike.
Assuntos
Anticorpos Neutralizantes/sangue , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Testes de Neutralização/métodos , Receptores Virais/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/terapia , Vacinas contra COVID-19/imunologia , Humanos , Imunização , Imunização Passiva , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Camundongos , Domínios Proteicos/imunologia , Soroterapia para COVID-19RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.
Assuntos
Anticorpos Neutralizantes/análise , Testes de Neutralização , SARS-CoV-2/isolamento & purificação , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , Estudos de Coortes , Humanos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and has led to calls for diagnostic tools to monitor and understand the transmission, pathogenesis, and epidemiology, as well as to evaluate future vaccination strategies. In this study, we have developed novel, to our knowledge, flexible ELISA-based assays for specific detection of human SARS-CoV-2 Abs against the receptor-binding domain, including an Ag sandwich ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the Ab responses to different areas on protein N and S and showed that the IgM, A, and G Ab responses against receptor-binding domain are significantly correlated to the disease severity. These assays and the data generated from them are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.
Assuntos
Anticorpos Antivirais/imunologia , COVID-19/diagnóstico , COVID-19/imunologia , Convalescença , SARS-CoV-2/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Índice de Gravidade de Doença , Adulto JovemRESUMO
No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP, have improved the elicitation of potent isolate-specific antibody responses in rabbits and macaques, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 µg ml-1. Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/isolamento & purificação , Bovinos/imunologia , HIV/imunologia , Imunização , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Células HEK293 , Proteína gp160 do Envelope de HIV/imunologia , HumanosRESUMO
Therapeutic proteins, including monoclonal antibodies, are typically manufactured using clonally derived, stable host cell lines, since consistent and predictable cell culture performance is highly desirable. However, selecting and preparing banks of stable clones takes considerable time, which inevitably extends overall development timelines for new therapeutics by delaying the start of subsequent activities, such as the scale-up of manufacturing processes. In the context of the coronavirus disease 2019 (COVID-19) pandemic, with its intense pressure for accelerated development strategies, we used a novel transposon-based Leap-In Transposase® system to rapidly generate high-titer stable pools and then used them directly for large scale-manufacturing of an anti-severe acute respiratory syndrome coronavirus 2 monoclonal antibody under cGMP. We performed the safety testing of our non-clonal cell bank, then used it to produce material at a 200L-scale for preclinical safety studies and formulation development work, and thereafter at 2000L scale for supply of material for a Phase 1 clinical trial. Testing demonstrated the comparability of critical product qualities between the two scales and, more importantly, that our final clinical trial product met all pre-set product quality specifications. The above expediated approach provided clinical trial material within 4.5 months, in comparison to 12-14 months for production of clinical trial material via the conventional approach.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Células CHO , COVID-19/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ensaios Clínicos Fase I como Assunto/métodos , Ensaios Clínicos Fase I como Assunto/normas , Cricetulus , Pandemias , Transposases , Carga ViralRESUMO
Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.
Assuntos
Desenho de Fármacos , Epitopos/química , Epitopos/imunologia , Estabilidade Proteica , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Macaca mulatta/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Testes de Neutralização , Conformação Proteica , Vírus Sinciciais Respiratórios/química , Vírus Sinciciais Respiratórios/imunologiaRESUMO
An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Desenho de Fármacos , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/genética , Infecções por HIV/imunologia , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , MutaçãoRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1005815.].
RESUMO
Challenge studies following passive immunization with neutralizing Abs suggest that an HIV vaccine could be efficacious were it able to elicit broadly neutralizing Abs (bNAbs). To better understand the requirements for activation of B cells producing bNAbs, we generated cell lines expressing bNAbs or their germline-reverted versions (gl-bNAbs) as BCRs. We then tested the abilities of the bNAb-expressing cells to recognize HIV pseudovirions and vaccine candidate proteins by binding and activation assays. The results suggest that HIV envelope (Env) Ag-expressing, infection-competent virions are poorly recognized by high-affinity bNAb-expressing cells, as measured by the inability of Ags to induce rapid increases in intracellular calcium levels. Other Ag forms appear to be highly stimulatory, in particular, soluble gp140 trimers and a multimerized, scaffolded epitope protein. Virions failed to efficiently activate bNAb-expressing B cells owing to delayed or inefficient BCR recognition, most likely caused by the low density of Env spikes. Importantly, B cells carrying gl-bNAb BCRs were not stimulated by any of the tested vaccine candidates. These data provide insight into why many HIV immunogens, as well as natural HIV infections, fail to rapidly stimulate bNAb responses and suggest that bNAb-expressing cell lines might be useful tools in evaluation of vaccine Ags for infectious diseases. Because soluble Env trimers or multimerized scaffolded epitopes are best at activating B cell-expressing bNAbs, these antigenic forms should be considered as preferred vaccine components, although they should be modified to better target naive gl-bNAb B cells.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Bioensaio/métodos , Técnicas Biossensoriais/métodos , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Animais , Antígenos HIV/imunologia , Humanos , Camundongos , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Eight of the 24 integrin heterodimers bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, and play essential roles in cell adhesion, migration, and homeostasis. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands including fibronectin, vitronectin, fibrinogen, nephronectin and the prodomain of the transforming growth factors to fulfill specific functions in cellular processes. Subtype-specific antibodies against RGD-binding integrins are desirable for investigating their specific functions. In this study, we discovered 11 antibodies that exhibit high specificity and affinity towards integrins αVß3, αVß5, αVß6, αVß8, and α5ß1 from a synthetic yeast-displayed Fab library. Of these, 6 are function-blocking antibodies containing an R(G/L/T) D motif in their CDR3 sequences. We report antibody binding specificity, kinetics, and binding affinity for purified integrin ectodomains as well as intact integrins on the cell surface. We further employed these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6=ß8>ß3>ß1=ß5.
RESUMO
Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVß3, αVß5, αVß6, αVß8, and α5ß1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6 = ß8 > ß3 > ß1 = ß5.
Assuntos
Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Cadeias beta de Integrinas/imunologia , Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/metabolismo , Cadeias beta de Integrinas/genética , Integrina alfaV/imunologia , Integrina alfaV/metabolismo , Integrinas/imunologia , Integrinas/metabolismo , Biblioteca de Peptídeos , Técnicas de Visualização da Superfície Celular , Ligação Proteica , Especificidade de AnticorposRESUMO
Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody-based universal antivenom to treat snakebite envenoming.
Assuntos
Antivenenos , Mordeduras de Serpentes , Humanos , Animais , Camundongos , Antivenenos/química , Mordeduras de Serpentes/tratamento farmacológico , Neurotoxinas/toxicidade , Anticorpos Amplamente Neutralizantes , Venenos de SerpentesRESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing antibody that neutralizes one virus but not a related virus. Through a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1-neutralizing antibody, to bind to the receptor binding domain of SARS-CoV-2 with >1000-fold increased affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of engineered CR3022 elucidated the molecular mechanisms by which the antibody can accommodate sequence differences in the epitopes between SARS-CoV-1 and SARS-CoV-2. This workflow provides a blueprint for the rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , SARS-CoV-2/genética , COVID-19/prevenção & controle , Anticorpos Antivirais , Testes de Neutralização , Anticorpos NeutralizantesRESUMO
Background: HIV infection dysregulates the B cell compartment, affecting memory B cell formation and the antibody response to infection and vaccination. Understanding the B cell response to SARS-CoV-2 in people living with HIV (PLWH) may explain the increased morbidity, reduced vaccine efficacy, reduced clearance, and intra-host evolution of SARS-CoV-2 observed in some HIV-1 coinfections. Methods: We compared B cell responses to COVID-19 in PLWH and HIV negative (HIV-ve) patients in a cohort recruited in Durban, South Africa, during the first pandemic wave in July 2020 using detailed flow cytometry phenotyping of longitudinal samples with markers of B cell maturation, homing, and regulatory features. Results: This revealed a coordinated B cell response to COVID-19 that differed significantly between HIV-ve and PLWH. Memory B cells in PLWH displayed evidence of reduced germinal centre (GC) activity, homing capacity, and class-switching responses, with increased PD-L1 expression, and decreased Tfh frequency. This was mirrored by increased extrafollicular (EF) activity, with dynamic changes in activated double negative (DN2) and activated naïve B cells, which correlated with anti-RBD-titres in these individuals. An elevated SARS-CoV-2-specific EF response in PLWH was confirmed using viral spike and RBD bait proteins. Conclusions: Despite similar disease severity, these trends were highest in participants with uncontrolled HIV, implicating HIV in driving these changes. EF B cell responses are rapid but give rise to lower affinity antibodies, less durable long-term memory, and reduced capacity to adapt to new variants. Further work is needed to determine the long-term effects of HIV on SARS-CoV-2 immunity, particularly as new variants emerge. Funding: This work was supported by a grant from the Wellcome Trust to the Africa Health Research Institute (Wellcome Trust Strategic Core Award [grant number 201433/Z/16/Z]). Additional funding was received from the South African Department of Science and Innovation through the National Research Foundation (South African Research Chairs Initiative [grant number 64809]), and the Victor Daitz Foundation.
Assuntos
COVID-19 , Infecções por HIV , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , África do Sul , Anticorpos AntiviraisRESUMO
The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we used a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduces the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity-matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for antiviral indications would benefit from affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.