RESUMEN
The ultimate vaccine against infections caused by Nipah virus should be capable of providing protection at the respiratory tractâthe most probable port of entry for this pathogen. Intranasally delivered vaccines, which target nasal-associated lymphoid tissue and induce both systemic and mucosal immunity, are attractive candidates for enabling effective vaccination against this lethal disease. Herein, the water-soluble polyphosphazene delivery vehicle assembles into nanoscale supramolecular constructs with the soluble extracellular portion of the Hendra virus attachment glycoproteinâa promising subunit vaccine antigen against both Nipah and Hendra viruses. These supramolecular constructs signal through Toll-like receptor 7/8 and promote binding interactions with mucinâan important feature of effective mucosal adjuvants. High mass contrast of phosphorus-nitrogen backbone of the polymer enables a successful visualization of nanoconstructs in their vitrified state by cryogenic electron microscopy. Here, we characterize the self-assembly of polyphosphazene macromolecule with biologically relevant ligands by asymmetric flow field flow fractionation, dynamic light scattering, fluorescence spectrophotometry, and turbidimetric titration methods. Furthermore, a polyphosphazene-enabled intranasal Nipah vaccine candidate demonstrates the ability to induce immune responses in hamsters and shows superiority in inducing total IgG and neutralizing antibodies when benchmarked against the respective clinical stage alum adjuvanted vaccine. The results highlight the potential of polyphosphazene-enabled nanoassemblies in the development of intranasal vaccines.
Asunto(s)
Administración Intranasal , Virus Nipah , Compuestos Organofosforados , Polímeros , Vacunas de Subunidad , Vacunas Virales , Compuestos Organofosforados/química , Compuestos Organofosforados/administración & dosificación , Polímeros/química , Virus Nipah/inmunología , Animales , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/química , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/química , Vacunas de Subunidad/administración & dosificación , Tamaño de la Partícula , Ensayo de Materiales , Materiales Biocompatibles/química , Nanopartículas/química , InmunizaciónRESUMEN
No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.
Asunto(s)
Infecciones por Henipavirus , Virus Nipah , Animales , Anticuerpos Monoclonales , Bangladesh , Chlorocebus aethiops , Glicoproteínas/metabolismo , Infecciones por Henipavirus/prevención & control , Primates , Ensayos Clínicos Fase I como AsuntoRESUMEN
The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.
Asunto(s)
Virus Hendra , Infecciones por Henipavirus , Virus Nipah , Humanos , Animales , Proteínas del Envoltorio Viral/genética , Virus Hendra/genética , Virus Nipah/genética , Glicoproteínas/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismoRESUMEN
Auro Vaccines LLC has developed a protein vaccine to prevent disease from Nipah and Hendra virus infection that employs a recombinant soluble Hendra glycoprotein (HeV-sG) adjuvanted with aluminum phosphate. This vaccine is currently under clinical evaluation in a Phase 1 study. The Benefit-Risk Assessment of VAccines by TechnolOgy Working Group (BRAVATO; ex-V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of protein vaccines. This will help key stakeholders to assess potential safety issues and understand the benefit-risk of such a vaccine platform. The structured and standardized assessment provided by the template may also help contribute to improved public acceptance and communication of licensed protein vaccines.
Asunto(s)
Virus Hendra , Infecciones por Henipavirus , Glicoproteínas , Infecciones por Henipavirus/prevención & control , Humanos , Medición de Riesgo , Vacunas SintéticasRESUMEN
Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses (HNVs) causing respiratory illness and severe encephalitis in humans, with fatality rates of 50-100%. There are no licensed therapeutics or vaccines to protect humans. HeV and NiV use a receptor-binding glycoprotein (G) and a fusion glycoprotein (F) to enter host cells. HNV F and G are the main targets of the humoral immune response, and the presence of neutralizing antibodies is a correlate of protection against NiV and HeV in experimentally infected animals. We describe here two cross-reactive F-specific antibodies, 1F5 and 12B2, that neutralize NiV and HeV through inhibition of membrane fusion. Cryo-electron microscopy structures reveal that 1F5 and 12B2 recognize distinct prefusion-specific, conserved quaternary epitopes and lock F in its prefusion conformation. We provide proof-of-concept for using antibody cocktails for neutralizing NiV and HeV and define a roadmap for developing effective countermeasures against these highly pathogenic viruses.
Asunto(s)
Anticuerpos Antivirales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Virus Hendra/inmunología , Virus Nipah/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Células CHO , Cricetulus , Reacciones Cruzadas , Células HEK293 , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/prevención & control , Humanos , Ratones , Internalización del VirusRESUMEN
Nipah and Hendra viruses are highly pathogenic bat-borne paramyxoviruses recently included in the WHO Blueprint priority diseases list. A fully registered horse anti-Hendra virus subunit vaccine has been in use in Australia since 2012. Based on the same immunogen, the Hendra virus attachment glycoprotein ectodomain, a subunit vaccine formulation for use in people is now in a Phase I clinical trial. We report that a single dose vaccination regimen of this human vaccine formulation protects against otherwise lethal challenges of either Hendra or Nipah virus in a nonhuman primate model. The protection against the Nipah Bangladesh strain begins as soon as 7 days post immunization with low dose of 0.1 mg protein subunit. Our data suggest this human vaccine could be utilized as efficient emergency vaccine to disrupt potential spreading of Nipah disease in an outbreak setting.
RESUMEN
Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 µg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Expresión Génica , Anticuerpos Anti-VIH/inmunología , Lactobacillus/metabolismo , Proteínas Recombinantes/inmunología , Anticuerpos Neutralizantes/genética , Transmisión de Enfermedad Infecciosa/prevención & control , Femenino , Anticuerpos Anti-VIH/genética , Infecciones por VIH/prevención & control , VIH-1 , Humanos , Lactobacillus/genética , Proteínas Recombinantes/genética , Vagina/inmunología , Vagina/microbiologíaRESUMEN
Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African green monkeys (AGMs) with HeV was recently demonstrated, and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen, and brain. Here, we demonstrate that m102.4, a potent HeV-neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease after infection with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV, and 12 subjects were infused twice with a 100-mg dose of m102.4 beginning at either 10, 24, or 72 hours after infection and again about 48 hours later. The presence of viral RNA, infectious virus, and HeV-specific immune responses demonstrated that all subjects were infected after challenge. All 12 AGMs that received m102.4 survived infection, whereas the untreated control subjects succumbed to disease on day 8 after infection. Animals in the 72-hour treatment group exhibited neurological signs of disease, but all animals started to recover by day 16 after infection. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Virus Hendra/patogenicidad , Infecciones por Henipavirus/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacocinética , Chlorocebus aethiops , Infecciones por Henipavirus/prevención & control , HumanosRESUMEN
Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.
Asunto(s)
Fármacos Anti-VIH/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Fragmentos de Péptidos/inmunología , Anticuerpos de Cadena Única/inmunología , Fármacos Anti-VIH/farmacología , Anticuerpos Neutralizantes/genética , Especificidad de Anticuerpos , Antígenos CD4/inmunología , Línea Celular , Relación Dosis-Respuesta Inmunológica , Epítopos/inmunología , Anticuerpos Anti-VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Anticuerpos de Cadena Única/genéticaRESUMEN
Antibodies can neutralize HIV-1 with potency and cross-reactivity that varies widely and is related but not correlated to their antigen-binding affinity. Therefore, in addition to measuring binding affinity, an evaluation of the antibody neutralizing activity in tissue cultures is important for development of antibody-based therapeutics, design of candidate vaccine immunogens, and understanding the mechanisms of virus entry, neutralization, and evasion of immune responses. The development of a standardized assay for measurement of the in vitro neutralizing activities of the antibody has remained a challenging goal in the last two decades. There are two types of widely used assays, which vary in details between different laboratories--assays based on cell line/pseudovirus and assays based on infection of peripheral blood mononuclear cells (PBMCs). Here we describe in detail the PBMC-based assay, which is more laborious but in our opinion represents a closer approximation of the in vivo situation. As with all other in vitro assays the results of such measurements are only an indication of the antibody potency in vivo, and animal studies and ultimately clinical trials are needed for the development of such antibodies as potential prophylactics and therapeutics.
Asunto(s)
Anticuerpos Antivirales/inmunología , VIH-1/inmunología , Pruebas de Neutralización/métodos , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/inmunología , VIH-1/aislamiento & purificación , Humanos , Región Variable de Inmunoglobulina/inmunología , Concentración 50 Inhibidora , Activación de Linfocitos , Linfocitos/virología , VolumetríaRESUMEN
BACKGROUND: Recently, several potently neutralizing fully human monoclonal antibodies (hmAbs) targeting the severe acute respiratory syndrome-associated coronavirus (SARS CoV) S glycoprotein, and the G glycoprotein of the paramyxoviruses Hendra virus (HeV) and Nipah virus (NiV) have been discovered [corrected]. OBJECTIVE: To examine, compare and contrast the functional characteristics of hmAbs with the potential for prophylaxis and treatment of diseases caused by SARS CoV, HeV and NiV. METHODS: A review of relevant literature. RESULTS/CONCLUSIONS: Structural, functional and biochemical analyses [corrected] have provided insights into the molecular mechanisms of receptor recognition and antibody neutralization, and suggested that these antibodies alone or in combination could fight the viruses' heterogeneity and mutability, which is a major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Coronavirus/inmunología , Virus Hendra/inmunología , Virus Nipah/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , HumanosRESUMEN
We have previously identified neutralizing human monoclonal antibodies against Nipah virus (NiV) and Hendra virus (HeV) by panning a large nonimmune antibody library against a soluble form of the HeV attachment-envelope glycoprotein G (sG HeV). One of these antibodies, m102, which exhibited the highest level of cross-reactive neutralization of both NiV and HeV G, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavy-chain variable domain and panning against sGHeV. One of the selected antibody Fab clones, m102.4, had affinity of binding to sGHeV that was equal to or higher than that of the other Fabs; it was converted to IgG1 and tested against infectious NiV and HeV. It exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 microg/mL, respectively. The virus-neutralizing activity correlated with the binding affinity of the antibody to sG HeV and sG NiV. m102.4 bound a soluble form of NiV G (sG NiV) better than it bound sG HeV, and it neutralized NiV better than HeV, despite being originally selected against sG HeV. These results suggest that m102.4 has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. It could be also used for prophylaxis and diagnosis, and as a research reagent.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Virus Hendra/fisiología , Infecciones por Henipavirus/inmunología , Virus Nipah/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Línea Celular Tumoral , Chlorocebus aethiops , Reacciones Cruzadas , Epítopos/inmunología , Glioblastoma , Semivida , Células HeLa , Virus Hendra/inmunología , Infecciones por Henipavirus/virología , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Pruebas de Neutralización , Virus Nipah/inmunología , Células Vero , Proteínas del Envoltorio Viral/inmunologíaAsunto(s)
Vacunas contra el SIDA/inmunología , VIH/química , VIH/fisiología , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas del Envoltorio Viral/química , Internalización del Virus , Secuencia de Aminoácidos , VIH/inmunología , Humanos , Fusión de Membrana , Datos de Secuencia Molecular , Unión Proteica , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Elicitation of broadly cross-reactive neutralizing antibodies (bcnAbs) in HIV infections is rare. To test the hypothesis that such antibodies could be elicited by HIV envelope glycoproteins (Envs) with unusual immunogenic properties and to identify novel bcnAbs, we used a soluble Env ectodomain (gp140) from a donor (R2) with high level of bcnAbs as an antigen for panning of an immune phage-displayed antibody library. The panning with the R2 Env resulted in significantly higher number of cross-reactive antibody clones than by using Envs from two other isolates (89.6 and IIIB). Two of the identified human monoclonal antibodies (hmAbs), m22 and m24, had sequences, neutralizing and binding activities similar or identical to those of the gp120-specific bcnAbs m18 and m14. The use of the R2 Env but not other Envs for panning resulted in the identification of a novel gp41-specific hmAb, m46. For several of the tested HIV-1 primary isolates its potency on molar basis was comparable to that of T20. It inhibited entry of primary isolates from different clades with an increased activity for cell lines with low CCR5 surface concentrations. The m46 neutralizing activity against a panel of clade C isolates was significantly higher in an assay based on peripheral blood mononuclear cells (4 out of 5 isolates were neutralized with an IC(50) in the range from 1.5 to 25 microg/ml) than in an assay based on a cell line with relatively high concentration of cell-surface-associated CCR5. In contrast to 2F5 and Z13, this antibody did not bind to denatured gp140 and gp41-derived peptides indicating a conformational nature of its epitope. It bound to a 5-helix bundle but not to N-heptad repeat coiled coils and a 6-helix bundle construct indicating contribution of both gp41 heptad repeats to its epitope and to a possible mechanism of neutralization. These results indicate that the R2 Env may contain unique exposed conserved epitopes that could contribute to its ability to elicit broadly cross-reactive antibodies in animals and humans; the newly identified antibodies may help in the development of novel vaccine immunogens and therapeutics.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas/inmunología , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Biblioteca de Péptidos , Anticuerpos Monoclonales/aislamiento & purificación , Unión Competitiva , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/clasificación , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Pruebas de Neutralización , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
The membrane-proximal external region (MPER) of HIV-1 gp41 is highly conserved and critical for the fusogenic ability of the virus. However, little is known about the activity of this region in the context of viral fusion. In this study we investigate the temporal exposure of MPER during the course of HIV-1 Env-mediated fusion. We employed the broadly neutralizing monoclonal antibodies 2F5 and 4E10, whose epitopes localize to this region as indicators for accessibility to this region. Time of addition experiments indicated that escape of HIV-1 infection inhibition by 2F5 and 4E10 occurred concomitantly with that of C34, a peptide that blocks the six-helix bundle formation and fusion, which was about 20 min later than escape of inhibition by the mAb b12 that blocks CD4-gp120 attachment. We also probed accessibility of the MPER region on fusion intermediates by measuring the binding of the monoclonal antibodies at different time points during the fusion reaction. Immunofluorescence and in-cell Western assays showed that binding of 2F5 and 4E10 decreased upon triggering HIV-1 Env-expressing cells with appropriate target cells. Addition of C34 did not counteract the loss of antibody binding, suggesting that changes in exposure of MPER occur independently of six-helix bundle formation.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/fisiología , VIH-1/patogenicidad , Fusión de Membrana , Anticuerpos Monoclonales , Reacciones Antígeno-Anticuerpo , Antígenos Virales , Proteína gp41 de Envoltorio del VIH/inmunología , Factores de Tiempo , Acoplamiento Viral , Internalización del VirusRESUMEN
It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays [J.M. Binley, T. Wrin, B. Korber, M.B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C.J. Petropoulos, D.R. Burton, Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies, J. Virol. 78 (2004) 13232-13252]. It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocytes compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 10(3) per cell) compared to cell lines with high CCR5 concentration (about 10(4) or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches.
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Anticuerpos Anti-VIH/fisiología , Infecciones por VIH/inmunología , VIH-1/inmunología , Receptores CCR5/metabolismo , Línea Celular , Infecciones por VIH/metabolismo , Células HeLa , Humanos , Pruebas de NeutralizaciónRESUMEN
The demand for new treatment options against HIV is becoming increasingly desperate as the side effects and the expansion and spread of drug-resistant virus within the infected population limit the clinical benefits provided by available anti-HIV drugs. Preparations of polyclonal antibodies have a long history of proven clinical utility against some viruses; however, they have enjoyed very limited success against HIV. Recent clinical trials and in vitro experiments suggest that monoclonal antibodies against HIV may have promise clinically. These antibodies and antibody-based reagents target either the viral envelope glycoprotein, the receptor (CD4) or coreceptor (CCR5) molecules, or transition-state structures that appear during viral entry. The challenge is whether an antibody-based therapy can be identified (with or without their small molecule brethren) that presents long-term clinical efficacy, low toxicity and minimal risk of clinical failure from viral resistance.
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Anticuerpos Anti-VIH/uso terapéutico , Infecciones por VIH/terapia , VIH-1/inmunología , Inmunoterapia/tendencias , Animales , Antígenos CD4/genética , Antígenos CD4/inmunología , Ensayos Clínicos como Asunto , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Receptores CCR5/inmunologíaRESUMEN
Polyclonal antibodies have a century-old history of being effective against some viruses; recently, monoclonal antibodies (mAbs) have also shown success. The humanized mAb Synagis (palivizumab), which is still the only mAb against a viral disease approved by the US FDA, has been widely used as a prophylactic measure against respiratory syncytial virus infections in neonates and immunocompromised individuals. The first fully human mAbs against two other paramyxoviruses, Hendra and Nipah virus, which can cause high (up to 75%) mortality, were recently developed; one of them, m101, showed exceptional potency against infectious virus. In an amazing pace of research, several potent human mAbs targeting the severe acute respiratory syndrome coronavirus S glycoprotein that can affect infections in animal models have been developed months after the virus was identified in 2003. A potent humanized mAb with therapeutic potential was recently developed against the West Nile virus. The progress in developing neutralizing human mAbs against Ebola, Crimean-Congo hemorrhagic fever, vaccinia and other emerging and biodefense-related viruses is slow. A major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies, is the viruses' heterogeneity and mutability. A related problem is the low binding affinity of crossreactive antibodies able to neutralize a variety of primary isolates. Combinations of mAbs or mAbs with other drugs, and/or the identification of potent new mAbs and their derivatives that target highly conserved viral structures, which are critical for virus entry into cells, are some of the possible solutions to these problems, and will continue to be a major focus of antiviral research.
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Anticuerpos Monoclonales , Anticuerpos Antivirales , Bioterrorismo/prevención & control , Enfermedades Transmisibles Emergentes/prevención & control , Virus/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Humanos , Pruebas de Neutralización , Virosis/prevención & control , Virus/genéticaRESUMEN
Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naïve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 microg/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 microg/ml, and 98% neutralization required only 1.6 microg/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.