RESUMO
Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Linfócitos B/imunologia , Epitopos de Linfócito B , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Memória Imunológica , Virus da Influenza A Subtipo H5N1/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Adulto JovemRESUMO
Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Linfócitos B/imunologia , Linhagem Celular , Células HEK293 , Infecções por HIV/imunologia , Humanos , Leucócitos Mononucleares , Estudos LongitudinaisRESUMO
Detailed studies of the broadly neutralizing antibodies (bNAbs) that underlie the best available examples of the humoral immune response to HIV are providing important information for the development of therapies and prophylaxis for HIV-1 infection. Here, we report a CD4-binding site (CD4bs) antibody, named N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. N6 evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain. In addition, structural analysis revealed that the orientation of N6 permitted it to avoid steric clashes with glycans, which is a common mechanism of resistance. Thus, an HIV-1-specific bNAb can achieve potent, near-pan neutralization of HIV-1, making it an attractive candidate for use in therapy and prophylaxis.
Assuntos
Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Especificidade de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Separação Celular , Proteína gp120 do Envelope de HIV/imunologia , HumanosRESUMO
Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominant mechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV , HIV-1/imunologia , Receptores de IgG/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células Cultivadas , Modelos Animais de Doenças , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência SímiaRESUMO
UNLABELLED: Extraordinary antibodies capable of near pan-neutralization of HIV-1 have been identified. One of the broadest is antibody 10E8, which recognizes the membrane-proximal external region (MPER) of the HIV-1 envelope and neutralizes >95% of circulating HIV-1 strains. If delivered passively, 10E8 might serve to prevent or treat HIV-1 infection. Antibody 10E8, however, is markedly less soluble than other antibodies. Here, we describe the use of both structural biology and somatic variation to develop optimized versions of 10E8 with increased solubility. From the structure of 10E8, we identified a prominent hydrophobic patch; reversion of four hydrophobic residues in this patch to their hydrophilic germ line counterparts resulted in an â¼10-fold decrease in turbidity. We also used somatic variants of 10E8, identified previously by next-generation sequencing, to optimize heavy and light chains; this process yielded several improved variants. Of these, variant 10E8v4 with 26 changes versus the parent 10E8 was the most soluble, with a paratope we showed crystallographically to be virtually identical to that of 10E8, a potency on a panel of 200 HIV-1 isolates also similar to that of 10E8, and a half-life in rhesus macaques of â¼10 days. An anomaly in 10E8v4 size exclusion chromatography that appeared to be related to conformational isomerization was resolved by engineering an interchain disulfide. Thus, by combining a structure-based approach with natural variation in potency and solubility from the 10E8 lineage, we successfully created variants of 10E8 which retained the potency and extraordinary neutralization breadth of the parent 10E8 but with substantially increased solubility. IMPORTANCE: Antibody 10E8 could be used to prevent HIV-1 infection, if manufactured and delivered economically. It suffers, however, from issues of solubility, which impede manufacturing. We hypothesized that the physical characteristic of 10E8 could be improved through rational design, without compromising breadth and potency. We used structural biology to identify hydrophobic patches on 10E8, which did not appear to be involved in 10E8 function. Reversion of hydrophobic residues in these patches to their hydrophilic germ line counterparts increased solubility. Next, clues from somatic variants of 10E8, identified by next-generation sequencing, were incorporated. A combination of structure-based design and somatic variant optimization led to 10E8v4, with substantially improved solubility and similar potency compared to the parent 10E8. The cocrystal structure of antibody 10E8v4 with its HIV-1 epitope was highly similar to that with the parent 10E8, despite 26 alterations in sequence and substantially improved solubility. Antibody 10E8v4 may be suitable for manufacturing.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/química , HIV-1/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Técnicas de Química Analítica , Cristalografia por Raios X , Dissulfetos , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Meia-Vida , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interações Hidrofóbicas e Hidrofílicas , Macaca mulatta , Modelos Moleculares , SolubilidadeRESUMO
UNLABELLED: The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55-62, 2014, http://dx.doi.org/10.1038/nature13036). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 µg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. IMPORTANCE: Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 µg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.
Assuntos
Anticorpos Neutralizantes/imunologia , Reações Cruzadas , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Mapeamento de Epitopos , Feminino , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.
Assuntos
Anticorpos Neutralizantes , Anticorpos Anti-HIV , HIV-1 , Macaca mulatta , Receptores de IgG , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , HIV-1/imunologia , Vírus da Imunodeficiência Símia/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Anticorpos Monoclonais/imunologia , Linfonodos/imunologia , Linfócitos T CD8-Positivos/imunologia , Afinidade de Anticorpos/imunologia , NF-kappa B/metabolismo , NF-kappa B/imunologia , Humanos , Infecções por HIV/imunologia , Infecções por HIV/virologia , Células Matadoras Naturais/imunologia , Anticorpos Amplamente Neutralizantes/imunologiaRESUMO
Background: There are limited global data on head-to-head comparisons of vaccine platforms assessing both humoral and cellular immune responses, stratified by pre-vaccination serostatus. The COVID-19 vaccination drive for the Indian population in the age group 18-45 years began in April 2021 when seropositivity rates in the general population were rising due to the delta wave of COVID-19 pandemic during April-May 2021. Methods: Between June 30, 2021, and Jan 28, 2022, we enrolled 691 participants in the age group 18-45 years across four clinical sites in India. In this non-randomised and laboratory blinded study, participants received either two doses of Covaxin® (4 weeks apart) or two doses of Covishield™ (12 weeks apart) as per the national vaccination policy. The primary outcome was the seroconversion rate and the geometric mean titre (GMT) of antibodies against the SARS-CoV-2 spike and nucleocapsid proteins post two doses. The secondary outcome was the frequency of cellular immune responses pre- and post-vaccination. Findings: When compared to pre-vaccination baseline, both vaccines elicited statistically significant seroconversion and binding antibody levels in both seronegative and seropositive individuals. In the per-protocol cohort, Covishield™ elicited higher antibody responses than Covaxin® as measured by seroconversion rate (98.3% vs 74.4%, p < 0.0001 in seronegative individuals; 91.7% vs 66.9%, p < 0.0001 in seropositive individuals) as well as by anti-spike antibody levels against the ancestral strain (GMT 1272.1 vs 75.4 binding antibody units/ml [BAU/ml], p < 0.0001 in seronegative individuals; 2089.07 vs 585.7 BAU/ml, p < 0.0001 in seropositive individuals). As participants at all clinical sites were not recruited at the same time, site-specific immunogenicity was impacted by the timing of vaccination relative to the delta and omicron waves. Surrogate neutralising antibody responses against variants-of-concern including delta and omicron was higher in Covishield™ recipients than in Covaxin® recipients; and in seropositive than in seronegative individuals after both vaccination and asymptomatic infection (omicron variant). T cell responses are reported from only one of the four site cohorts where the vaccination schedule preceded the omicron wave. In seronegative individuals, Covishield™ elicited both CD4+ and CD8+ spike-specific cytokine-producing T cells whereas Covaxin® elicited mainly CD4+ spike-specific T cells. Neither vaccine showed significant post-vaccination expansion of spike-specific T cells in seropositive individuals. Interpretation: Covishield™ elicited immune responses of higher magnitude and breadth than Covaxin® in both seronegative individuals and seropositive individuals, across cohorts representing the pre-vaccination immune history of most of the vaccinated Indian population. Funding: Corporate social responsibility (CSR) funding from Hindustan Unilever Limited (HUL) and Unilever India Pvt. Ltd. (UIPL).
RESUMO
We demonstrate that at least three different promoter variant strains of HIV-1 subtype C have been gradually expanding and replacing the standard subtype C viruses in India, and possibly in South Africa and other global regions, over the past decade. The new viral strains contain an additional NF-κB, NF-κB-like, or RBEIII site in the viral promoter. Although the acquisition of an additional RBEIII site is a property shared by all the HIV-1 subtypes, acquiring an additional NF-κB site remains an exclusive property of subtype C. The acquired κB site is genetically distinct, binds the p50-p65 heterodimer, and strengthens the viral promoter at the levels of transcription initiation and elongation. The 4-κB viruses dominate the 3-κB "isogenic" viral strains in pairwise competition assays in T-cell lines, primary cells, and the ecotropic human immunodeficiency virus mouse model. The dominance of the 4-κB viral strains is also evident in the natural context when the subjects are coinfected with κB-variant viral strains. The mean plasma viral loads, but not CD4 counts, are significantly different in 4-κB infection suggesting that these newly emerging strains are probably more infectious. It is possible that higher plasma viral loads underlie selective transmission of the 4-κB viral strains. Several publications previously reported duplication or deletion of diverse transcription factor-binding sites in the viral promoter. Unlike previous reports, our study provides experimental evidence that the new viral strains gained a potential selective advantage as a consequence of the acquired transcription factor-binding sites and importantly that these strains have been expanding at the population level.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , Repetição Terminal Longa de HIV , HIV-1/genética , NF-kappa B/metabolismo , Transcrição Gênica , Adulto , Estudos de Coortes , Feminino , Regulação Viral da Expressão Gênica , Infecções por HIV/genética , HIV-1/química , HIV-1/classificação , HIV-1/fisiologia , Humanos , Masculino , Dados de Sequência Molecular , NF-kappa B/genética , Ligação Proteica , Replicação Viral , Adulto JovemRESUMO
BACKGROUND & OBJECTIVES: The complementary and alternative medicines (CAM) have not been systematically evaluated for the management of HIV/AIDS patients. In a prospective, single-site, open-label, non-randomized, controlled, pilot trial, we evaluated a polyherbal formulation (PHF) for its safety and efficacy in treating subjects with HIV-AIDS. METHODS: A total of 32 and 31 subjects were enrolled under the PHF and highly active antiretroviral treatment (HAART) arms, respectively, and followed up for a period of 24 months. Plasma viral RNA, CD4 cell count and blood chemistry were monitored at 3-month intervals. Following mid-term safety evaluation, 12 subjects from the PHF arm were shifted to HAART and were followed separately as PHF-to-HAART arm, for the rest of the period. RESULTS: The HAART arm was characterized by significant improvements in CD4 cell count (154.4 cells/µl/year, P<0.001) and reduction in plasma viral load within 3 to 6 months (-0.431+ 0.004 log 10 IU/month, P<0.001). In contrast, the PHF arm showed a profile of CD4 cell loss at remarkably slower kinetics (14.3 cells/µl/year, P=0.021) and insignificant reduction in the viral load. The PHF and HAART arms did not differ significantly in the occurrence of AIDS-related illnesses over the study period of 24 months. In the PHF-to-HAART arm, the rates of CD4 count and reduction in viral load were significant and comparable to that of the HAART group. In the PHF arm, at 1 month, a significant increase in CD4 cell count and a concomitant decrease in viral load were seen. INTERPRETATION & CONCLUSIONS: The PHF appears to have provided protection by delaying the kinetics of CD4 cell reduction. Given the several study limitations, drawing assertive inferences from the data is challenging. Future studies with a stringent study design are warranted to confirm these findings.
Assuntos
Infecções por HIV/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Adulto , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/efeitos dos fármacos , Terapias Complementares , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Segurança do Paciente , Projetos Piloto , Estudos Prospectivos , RNA Viral/sangue , Resultado do Tratamento , Carga Viral/efeitos dos fármacosRESUMO
The amino-acid composition of the immunoglobulin variable region has been observed to impact antibody pharmacokinetics (PK). Here, we sought to improve the PK of the broad HIV-1-neutralizing VRC01-class antibodies, VRC07-523LS and N6LS, by reducing the net positive charge in their variable domains. We used a structure-guided approach to generate a panel of antibody variants incorporating select Arg or Lys substituted to Asp, Gln, Glu, or Ser. The engineered variants exhibited reduced affinity to heparin, reduced polyreactivity, and improved PK in human FcRn-transgenic mice. One variant, VRC07-523LS.v34, with three charge substitutions, had an observed in vivo half-life and an estimated human half-life of 10.8 and 60 days, respectively (versus 5.4 and 38 days for VRC07-523LS) and retained functionality, neutralizing 92% of a 208-strain panel at a geometric mean IC80 <1 µg/mL. Another variant, N6LS.C49, with two charge substitutions, had an observed in vivo half-life and an estimated human half-life of 14.5 and 80 days (versus 9.0 and 44 days for N6LS) and neutralized ~80% of 208 strains at a geometric mean IC80 <1 µg/mL. Since Arg and Lys residues are prevalent in human antibodies, we propose substitution of select Arg or Lys with Asp, Gln, Glu, or Ser in the framework region as a general means to improve PK of therapeutic antibodies.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Camundongos , Animais , Anticorpos Anti-HIV , Anticorpos Amplamente Neutralizantes , Camundongos Transgênicos , Infecções por HIV/tratamento farmacológico , Anticorpos NeutralizantesRESUMO
Detailed characterisation of immune responses induced by COVID-19 vaccines rolled out in India: COVISHIELDTM (CS) and COVAXIN® (CO) in a pre-exposed population is only recently being discovered. We addressed this issue in subjects who received their primary series of vaccination between November 2021 and January 2022. Both vaccines are capable of strongly boosting Wuhan Spike-specific neutralising antibody, polyfunctional Th1 cytokine producing CD4+ T-cells and single IFN-γ + CD8+ T-cells. Consistent with inherent differences in vaccine platform, the vector-based CS vaccine-induced immunity was of greater magnitude, breadth, targeting Delta and Omicron variants compared to the whole-virion inactivated vaccine CO, with CS vaccinees showing persistent CD8+ T-cells responses until 3 months post primary vaccination. This study provides detailed evidence on the magnitude and quality of CS and CO vaccine induced responses in subjects with pre-existing SARS-CoV-2 immunity in India, thereby mitigating vaccine hesitancy arguments in such a population, which remains a global health challenge.
RESUMO
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.
Assuntos
Anticorpos Biespecíficos , Infecções por HIV , HIV-1 , Humanos , Animais , Camundongos , Anticorpos Neutralizantes , Testes de Neutralização , Anticorpos Anti-HIV , Sítios de LigaçãoRESUMO
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.
Assuntos
Infecções por HIV , HIV-1 , Animais , Anticorpos Amplamente Neutralizantes , Meia-Vida , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Peptídeo Hidrolases , AminoácidosRESUMO
Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here, a trispecific bNAb reduces viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounds transiently and returns to low levels, through CD8-mediated immune control. These viruses remain sensitive to the trispecific antibody, despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppresses the emergence of resistance in viruses derived from HIV-1-infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 neutralizing antibodies, therefore, mediate potent antiviral activity in vivo and may minimize the potential for immune escape.
Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/uso terapêutico , Anticorpos Anti-HIV/uso terapêutico , Evasão da Resposta Imune/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Vírus da Imunodeficiência Símia/imunologia , Animais , Antivirais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Humanos , Imunoterapia/métodos , Macaca mulatta , Células THP-1 , Viremia/prevenção & controle , Viremia/terapiaRESUMO
Primary HIV-1 infection can be classified into six Fiebig stages based on virological and serological laboratory testing, whereas simian-HIV (SHIV) infection in nonhuman primates (NHPs) is defined in time post-infection, making it difficult to extrapolate NHP experiments to the clinics. We identified and extensively characterized the Fiebig-equivalent stages in NHPs challenged intrarectally or intravenously with SHIVAD8-EO. During the first month post-challenge, intrarectally challenged monkeys were up to 1 week delayed in progression through stages. However, regardless of the challenge route, stages I-II predominated before, and stages V-VI predominated after, peak viremia. Decrease in lymph node (LN) CD4+ T cell frequency and rise in CD8+ T cells occurred at stage V. LN virus-specific CD8+ T cell responses, dominated by degranulation and TNF, were first detected at stage V and increased at stage VI. A similar late elevation in follicular CXCR5+ CD8+ T cells occurred, consistent with higher plasma CXCL13 levels at these stages. LN SHIVAD8-EO RNA+ cells were present at stage II, but appeared to decline at stage VI when virions accumulated in follicles. Fiebig-equivalent staging of SHIVAD8-EO infection revealed concordance of immunological events between intrarectal and intravenous infection despite different infection progressions, and can inform comparisons of NHP studies with clinical data.
Assuntos
Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Administração Intravenosa , Administração Retal , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Progressão da Doença , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1 , Humanos , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/classificação , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Fatores de Tempo , Pesquisa Translacional Biomédica , Carga Viral , Viremia/imunologia , Viremia/virologiaRESUMO
Passive transfer of broadly neutralizing antibodies is showing promise in the treatment and prevention of HIV-1. One class of antibodies, the VRC01 class, appears especially promising. To improve VRC01-class antibodies, we combined structure-based design with a matrix-based approach to generate VRC01-class variants that filled an interfacial cavity, used diverse third-complementarity-determining regions, reduced potential steric clashes, or exploited extended contacts to a neighboring protomer within the envelope trimer. On a 208-strain panel, variant VRC01.23LS neutralized 90% of the panel at a geometric mean IC80 less than 1 µg/ml, and in transgenic mice with human neonatal-Fc receptor, the serum half-life of VRC01.23LS was indistinguishable from that of the parent VRC01LS, which has a half-life of 71 d in humans. A cryo-electron microscopy structure of VRC01.23 Fab in complex with BG505 DS-SOSIP.664 Env trimer determined at 3.4-Å resolution confirmed the structural basis for its ~10-fold improved potency relative to VRC01. Another variant, VRC07-523-F54-LS.v3, neutralized 95% of the 208-isolated panel at a geometric mean IC80 of less than 1 µg/ml, with a half-life comparable to that of the parental VRC07-523LS. Our matrix-based structural approach thus enables the engineering of VRC01 variants for HIV-1 therapy and prevention with improved potency, breadth, and pharmacokinetics.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Infecções por HIV , HIV-1/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/farmacologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Camundongos KnockoutRESUMO
CIS43 is a potent neutralizing human mAb that targets a highly conserved "junctional" epitope in the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). Enhancing the durability of CIS43 in vivo will be important for clinical translation. Here, 2 approaches were used to improve the durability of CIS43 in vivo while maintaining potent neutralization. First, the Fc domain was modified with the LS mutations (CIS43LS) to increase CIS43 binding affinity for the neonatal Fc receptor (FcRn). CIS43LS and CIS43 showed comparable in vivo protective efficacy. CIS43LS had 9- to 13-fold increased binding affinity for human (6.2 nM versus 54.2 nM) and rhesus (25.1 nM versus 325.8 nM) FcRn at endosomal pH 6.0 compared with CIS43. Importantly, the half-life of CIS43LS in rhesus macaques increased from 22 days to 39 days compared with CIS43. The second approach for sustaining antibody levels of CIS43 in vivo is through adeno-associated virus (AAV) expression. Mice administered once with AAV-expressing CIS43 had sustained antibody levels of approximately 300 µg/mL and mediated protection against sequential malaria challenges up to 36 weeks. Based on these data, CIS43LS has advanced to phase I clinical trials, and AAV delivery provides a potential next-generation approach for malaria prevention.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/genética , Anticorpos Antiprotozoários/administração & dosagem , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/genética , Dependovirus/genética , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Macaca mulatta , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Proteínas de Protozoários/imunologiaRESUMO
Broadly neutralizing antibodies are showing promise in the treatment and prevention of HIV-1, with several now being evaluated clinically. Some lead clinical candidates, including antibodies CAP256-VRC26.25, N6, PGT121, and VRC07-523, have one or more N-linked glycosylation sequons in their variable domains (Fvs) from somatic hypermutation, and these glycans increase chemical heterogeneity, complicating the manufacture of these antibodies as products. Here we propose a general method to remove Fv glycans and use this method to develop engineered versions of these four antibodies with Fv glycans removed. When germline residues were introduced to remove each glycan, antibody properties between wild type and mutant were not significantly altered for CAP256-VRC26.25 and PGT121; however, germline mutants for N6 and VRC07-523 showed increased polyreactivity, which is known to correlate with unfavorable in vivo pharmacokinetics. To reduce polyreactivity induced by removal of Fv glycan, we mutated aromatic residues and arginines structurally proximal to the removed glycan and identified Fv glycan-removed variants with low polyreactivity for N6 and VRC07-523. Two such variants, N6-N72LCQ-R18LCD and VRC07-523-N72LCQ-R24LCD, showed thermostability, neutralization potency and breadth, and half-life in humanized FcRn mice that were similar to their wild-type Fv-glycosylated counterparts. The removal of Fv glycan and reduction of chemical heterogeneity were confirmed by liquid chromatography-mass spectrometry. With reduced heterogeneity, the Fv-glycan-removed variants developed here may have utility as products for treating or preventing infection by HIV-1.
Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1 , Região Variável de Imunoglobulina/imunologia , Animais , Glicosilação , Anticorpos Anti-HIV/química , Infecções por HIV/prevenção & controle , Humanos , Região Variável de Imunoglobulina/química , Camundongos , PolissacarídeosRESUMO
Broadly neutralizing antibodies (bNAbs) represent a promising alternative to antiretroviral drugs for HIV-1 prevention and treatment. Selected antibodies to the CD4-binding site bolster envelope trimer binding via quaternary contacts. Here, we rationally engraft a new paratope, i.e., the extended heavy-chain framework region 3 (FR3) loop of VRC03, which mediates quaternary interaction, onto several potent bNAbs, enabling them to reach an adjacent gp120 protomer. The interactive quaternary surface is delineated by solving the crystal structure of two FR3 loop-chimeric antibodies. Chimerization enhances the neutralizing activity of several potent bNAbs against a majority of global HIV-1 strains. Compared to unmodified antibodies, chimeric antibodies display lower autoreactivity and prolonged in vivo half-life in huFcRn mice and rhesus macaques. Thus, paratope engraftment may be used to expand the epitope repertory of natural antibodies, improving their functionality for disease prevention and treatment.