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1.
Immunology ; 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38952142

RESUMEN

Targeting immune receptors on T cells is a common strategy to treat cancer and autoimmunity. Frequently, this is accomplished through monoclonal antibodies targeting the ligand binding sites of stimulatory or inhibitory co-receptors. Blocking ligand binding prevents downstream signalling and modulates specific T cell functions. Since 1985, the FDA has approved over 100 monoclonal antibodies against immune receptors. This therapeutic approach significantly improved the care of patients with numerous immune-related conditions; however, many patients are unresponsive, and some develop immune-related adverse events. One reason for that is the lack of consideration for the localization of these receptors on the cell surface of the immune cells in the context of the immune synapse. In addition to blocking ligand binding, changing the location of these receptors on the cell surface within the different compartments of the immunological synapse could serve as an alternative, efficient, and safer approach to treating these patients. This review discusses the potential therapeutic advantages of altering proteins' localization within the immune synapse and summarizes published work in this field. It also discusses the novel use of bispecific antibodies to induce the clustering of receptors on the cell surface. It presents the rationale for developing novel antibodies, targeting the organization of signalling receptor complexes on the cell surface. This approach offers an innovative and emerging technology to treat cancer patients resistant to current immunotherapies.

2.
Proc Natl Acad Sci U S A ; 110(33): 13540-5, 2013 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-23878231

RESUMEN

In the absence of an effective HIV-1 vaccine, passive immunization using broadly neutralizing Abs or Ab-like molecules could provide an alternative to the daily administration of oral antiretroviral agents that has recently shown promise as preexposure prophylaxis. Currently, no single broadly neutralizing Ab (bNAb) or combination of bNAbs neutralizes all HIV-1 strains at practically achievable concentrations in vivo. To address this problem, we created bispecific Abs that combine the HIV-1 inhibitory activity of ibalizumab (iMab), a humanized mAb directed to domain 2 of human CD4, with that of anti-gp120 bNAbs. These bispecific bNAbs (BibNAbs) exploit iMab's potent anti-HIV-1 activity and demonstrated clinical efficacy and safety to anchor and thereby concentrate a second broadly neutralizing agent at the site of viral entry. Two BibNabs, PG9-iMab and PG16-iMab, exhibit exceptional breadth and potency, neutralizing 100% of the 118 viruses tested at low picomolar concentrations, including viruses resistant to both parental mAbs. The enhanced potency of these BibNAbs was entirely dependent on CD4 anchoring, not on membrane anchoring per se, and required optimal Ab geometry and linker length. We propose that iMab-based BibNAbs, such as PG9-iMab and PG16-iMab, are promising candidates for passive immunization to prevent HIV-1 infection.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/prevención & control , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Inmunización Pasiva/métodos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Neutralizantes/farmacología , Antígenos CD4/inmunología , Cromatografía en Gel , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Anti-VIH/farmacología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Pruebas de Neutralización , Resonancia por Plasmón de Superficie
3.
Mol Ther Oncol ; 32(3): 200839, 2024 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-39072290

RESUMEN

Targeting immune checkpoint receptors on T cells is a common cancer treatment strategy. Frequently, this is accomplished through antibodies targeting the ligand of inhibitory co-receptors. Blocking the immune checkpoint PD-1 binding to its ligands PD-L1 and PD-L2 prevents downstream signaling and enhances anti-tumor T cell responses. This approach improves cancer patients' outcomes. However, only one-third of the patients respond to these treatments. To better understand the mechanism of anti-PD-1 antibodies, we explored the location of PD-1 within the immune synapse. Surprisingly, we discovered that anti-PD-1 antibodies, besides blocking the interaction between PD-1 and its ligands, also removed PD-1 from the synapse. We demonstrated a correlation between removing PD-1 from the synapse by anti-PD-1 antibodies and the extent of T cell activation. Interestingly, a short version of the anti-PD-1 antibody, F(ab')2, failed to remove PD-1 from the synapse and activate T cells. Using the syngeneic tumor model, we showed a superior anti-tumor effect of the anti-PD-1 antibody over the shorter version of the same antibody. Our data indicate that anti-PD-1 antibodies activate T cells by removing PD-1 from the synapse, and changing the location of PD-1 or other immune receptors within the immune synapse could serve as an alternative, efficient approach to treat cancer.

4.
Life Sci Alliance ; 6(2)2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36622343

RESUMEN

Signaling lymphocyte activation molecule family member 6 (SLAMF6) is a T cell co-receptor. Previously, we showed that SLAMF6 clustering was required for T cell activation. To better understand the relationship between SLAMF6 location and function and to evaluate the role of SLAMF6 as a therapeutic target, we investigated how its compartmentalization on the cell surface affects T cell functions. We used biochemical and co-culture assays to show that T cell activity is enhanced when SLAMF6 colocalizes with the CD3 complex. Mechanistically, co-immunoprecipitation analysis revealed the SLAMF6-interacting proteins to be those essential for signaling downstream of T cell receptor, suggesting the two receptors share downstream signaling pathways. Bispecific anti-CD3/SLAMF6 antibodies, designed to promote SLAMF6 clustering with CD3, enhanced T cell activation. Meanwhile, anti-CD45/SLAMF6 antibodies inhibited SLAMF6 clustering with T cell receptor, likely because of the steric hindrance, but nevertheless enhanced T cell activation. We conclude that SLAMF6 bispecific antibodies have a role in modulating T cell responses, and future work will evaluate the therapeutic potential in tumor models.


Asunto(s)
Receptores de Antígenos de Linfocitos T , Linfocitos T , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal
5.
bioRxiv ; 2023 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-38014028

RESUMEN

Targeting immune checkpoint receptors on T cells is a common cancer treatment strategy. Frequently, this is accomplished through antibodies targeting the ligand of inhibitory co-receptors. Blocking the immune checkpoint PD-1 binding to its ligands PD-L1 and PD-L2 prevents downstream signaling and enhances anti-tumor T cell responses. This approach improved cancer patients' outcome. However, only one-third of the patients respond to these treatments. To better understand the mechanism of anti-PD-1 antibodies, we explored the location of PD-1 within the immune synapse. Surprisingly, we discovered that anti-PD-1 antibodies, besides blocking the interaction between PD-1 and its ligands, also removed PD-1 from the synapse. We demonstrated a correlation between removing PD-1 from the synapse by anti-PD-1 antibodies and the extent of T cell activation. Interestingly, a short version of the anti-PD-1 antibody, F(ab') 2 , failed to remove PD-1 from the synapse and activate T cells. Using syngeneic tumor model, we showed a superior anti-tumor effect to anti-PD-1 antibody over the shorter version of the antibody. Our data indicates that anti-PD-1 antibodies activate T cells by removing PD-1 away from the synapse and changing the location of PD-1 or other immune receptors within immune synapse could serve as an alternative, efficient approach to treat cancer.

6.
J Virol ; 84(14): 6935-42, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20463063

RESUMEN

Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.


Asunto(s)
Fármacos Anti-VIH , Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Mapeo Epitopo , VIH-1/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Fármacos Anti-VIH/inmunología , Fármacos Anti-VIH/farmacología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/química , Antígenos CD4/genética , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Internalización del Virus
7.
J Virol ; 83(3): 1422-32, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19019970

RESUMEN

Human immunodeficiency virus clade C (HIV-C) accounts for >56% of all HIV infections worldwide. To investigate vaccine safety and efficacy in nonhuman primates, a pathogenic, R5-tropic, neutralization-sensitive simian-human immunodeficiency virus (SHIV) carrying HIV-C env would be desirable. We have constructed SHIV-2873Ni, an R5-tropic SHIV carrying a primary pediatric HIV-C env gene isolated from a 2-month-old Zambian infant, who died within 1 year of birth. SHIV-2873Ni was constructed using SHIV-1157ipd3N4 (R. J. Song, A. L. Chenine, R. A. Rasmussen, C. R. Ruprecht, S. Mirshahidi, R. D. Grisson, W. Xu, J. B. Whitney, L. M. Goins, H. Ong, P. L. Li, E. Shai-Kobiler, T. Wang, C. M. McCann, H. Zhang, C. Wood, C. Kankasa, W. E. Secor, H. M. McClure, E. Strobert, J. G. Else, and R. M. Ruprecht. J. Virol. 80:8729-8738, 2006) as the backbone, since the latter contains additional NF-kappaB sites in the long terminal repeats to enhance viral replicative capacity. The parental virus, SHIV-2873Ni, was serially passaged through five rhesus monkeys (RMs); SHIV-2873Nip, the resulting passaged virus, was reisolated from the fourth recipient about 1 year postinoculation. SHIV-2873Nip was replication competent in RM peripheral blood mononuclear cells of all random donors tested and was exclusively R5 tropic, and its env gene clustered with HIV-C by phylogenetic analysis; its moderate [corrected] sensitivity to neutralization led to classification as a tier 2 [corrected] virus. Indian-origin RMs were inoculated by different mucosal routes, resulting in high peak viral RNA loads. Signs of virus-induced disease include depletion of gut CD4(+) T lymphocytes, loss of memory T cells in blood, and thrombocytopenia that resulted in fatal cerebral hemorrhage. SHIV-2873Nip is a highly replication-competent, mucosally transmissible, pathogenic R5-tropic virus that will be useful to study viral pathogenesis and to assess the efficacy of immunogens targeting HIV-C Env.


Asunto(s)
Infecciones por VIH/virología , VIH/inmunología , Pruebas de Neutralización , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Secuencia de Bases , Cartilla de ADN , Progresión de la Enfermedad , Genes env , VIH/genética , VIH/aislamiento & purificación , VIH/fisiología , Humanos , Lactante , Macaca mulatta , Filogenia , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Replicación Viral
8.
Retrovirology ; 6: 65, 2009 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-19602283

RESUMEN

BACKGROUND: Although pig-tailed macaques (Macaca nemestrina) have been used in AIDS research for years, less is known about the early immunopathogenic events in this species, as compared to rhesus macaques (Macaca mulatta). Similarly, the events in early infection are well-characterized for simian immunodeficiency viruses (SIV), but less so for chimeric simian-human immunodeficiency viruses (SHIV), although the latter have been widely used in HIV vaccine studies. Here, we report the consequences of intrarectal infection with a CCR5-tropic clade C SHIV-1157ipd3N4 in pig-tailed macaques. RESULTS: Plasma and cell-associated virus was detectable in peripheral blood and intestinal tissues of all four pig-tailed macaques following intrarectal inoculation with SHIV-1157ipd3N4. We also observed a rapid and irreversible loss of CD4+ T cells at multiple mucosal sites, resulting in a marked decrease of CD4:CD8 T cell ratios 0.5-4 weeks after inoculation. This depletion targeted subsets of CD4+ T cells expressing the CCR5 coreceptor and having a CD28-CD95+ effector memory phenotype, consistent with the R5-tropism of SHIV-1157ipd3N4. All three animals that were studied beyond the acute phase seroconverted as early as week 4, with two developing cross-clade neutralizing antibody responses by week 24. These two animals also demonstrated persistent plasma viremia for >48 weeks. One of these animals developed AIDS, as shown by peripheral blood CD4+ T-cell depletion starting at 20 weeks post inoculation. CONCLUSION: These findings indicate that SHIV-1157ipd3N4-induced pathogenesis in pig-tailed macaques followed a similar course as SIV-infected rhesus macaques. Thus, R5 SHIV-C-infection of pig-tailed macaques could provide a useful and relevant model for AIDS vaccine and pathogenesis research.


Asunto(s)
VIH/crecimiento & desarrollo , VIH/inmunología , Macaca nemestrina/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Anticuerpos Antivirales/sangre , Sangre/virología , Antígenos CD28/análisis , Relación CD4-CD8 , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/inmunología , VIH/genética , Mucosa Intestinal/virología , Subgrupos Linfocitarios/inmunología , Pruebas de Neutralización , Receptores CCR5/análisis , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Receptor fas
9.
Retrovirology ; 5: 94, 2008 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-18928523

RESUMEN

BACKGROUND: Infection of nonhuman primates with simian immunodeficiency virus (SIV) or chimeric simian-human immunodeficiency virus (SHIV) strains is widely used to study lentiviral pathogenesis, antiviral immunity and the efficacy of AIDS vaccine candidates. SHIV challenges allow assessment of anti-HIV-1 envelope responses in primates. As such, SHIVs should mimic natural HIV-1 infection in humans and, to address the pandemic, encode HIV-1 Env components representing major viral subtypes worldwide. RESULTS: We have developed a panel of clade C R5-tropic SHIVs based upon env of a Zambian pediatric isolate of HIV-1 clade C, the world's most prevalent HIV-1 subtype. The parental infectious proviral clone, SHIV-1157i, was rapidly passaged through five rhesus monkeys. After AIDS developed in the first animal at week 123 post-inoculation, infected blood was infused into a sixth monkey. Virus reisolated at this late stage was still exclusively R5 tropic and mucosally transmissible. Here we describe the long-term follow-up of this initial cohort of six monkeys. Two have remained non-progressors, whereas the other four gradually progressed to AIDS within 123-270 weeks post-exposure. Two progressors succumbed to opportunistic infections, including a case of SV40 encephalitis. CONCLUSION: These data document the disease progression induced by the first mucosally transmissible, pathogenic R5 non-clade B SHIV and suggest that SHIV-1157i-derived viruses, including the late-stage, highly replication-competent SHIV-1157ipd3N4 previously described (Song et al., 2006), display biological characteristics that mirror those of HIV-1 clade C and support their expanded use for AIDS vaccine studies in nonhuman primates.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/transmisión , VIH-1/patogenicidad , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Proteínas del Envoltorio Viral/genética , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/patología , Síndrome de Inmunodeficiencia Adquirida/virología , Secuencia de Aminoácidos , Animales , Línea Celular , VIH-1/genética , VIH-1/fisiología , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Alineación de Secuencia , Pase Seriado , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Proteínas del Envoltorio Viral/metabolismo , Carga Viral , Replicación Viral
10.
Mol Ther ; 15(5): 1007-1015, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-28182892

RESUMEN

Transfection of DNA vaccines with chemokines may recruit dendritic cells (DCs) locally to capture the antigenic genes and their gene products to generate enhanced CD8+ cytotoxic T lymphocytes (CTLs). In this study, we investigated the effects of macrophage inflammatory protein (MIP)-1α, MIP-3α, and MIP-3ß on human immunodeficiency virus (HIV) Gag DNA vaccination. The chemokine plasmids markedly enhanced the local infiltration of inflammatory cells and increased the presence of CD11c+ B7.2+-activated DCs. MIP-1α and MIP-3α were potent adjuvants in augmenting CTLs and afforded strong protection to immunized animals against challenge with vaccinia virus expressing Gag (vv-Gag). However, decreased humoral response was observed. MIP-3ß plasmid did not dramatically alter immunity. The chemokine inoculation time with respect to DNA vaccine priming was also investigated. The injection of pMIP-3α three days before Gag plasmid (pGag) vaccination markedly increased specific CTLs compared with simultaneous injection and led to higher protection against vv-Gag. Immunity was also shifted toward a T-helper type-1 (Th1) response. In contrast, inoculation with pMIP-3α three days after pGag vaccination shifted immunity toward a Th2 response. Our data suggest that administration of a chemokine with DNA vaccines offers a valuable strategy to modulate the efficacy and polarization of specific immunity and that chemokine-antigen timing is critical in determining overall biological effects.

11.
AIDS ; 21(14): 1841-8, 2007 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-17721091

RESUMEN

OBJECTIVE: To determine whether multigenic protein immunogens including native, trimeric HIV clade C (HIV-C) gp160 could cross-protect macaques against mucosal challenge with clade C (SHIV-C) mismatched for env. DESIGN: Because AIDS vaccine recipients are unlikely to encounter exactly matched HIV strains and to represent the diversity of locally circulating HIV-C strains, we selected env genes to generate the gp160 immunogen and SHIV-C from different, recently infected infants of the same clinical cohort in Zambia. In a model of postnatal HIV-C transmission, infant macaques were immunized with soluble viral proteins, including trimeric HIV1084i Env, and challenged with SHIV-1157ip; protein-only vaccination was compared with a DNA prime/protein boost strategy. METHODS: All vaccinated and control monkeys were exposed orally to low-dose, R5-tropic SHIV-1157ip encoding heterologous env. Animals with no or only transient infection were rechallenged intrarectally with a high dose of R5 SHIV-1157ipd3N4, a 'late', animal-evolved SHIV-1157ip variant. Animals were followed prospectively for immune parameters and viral RNA loads. RESULTS: Vaccination induced cross-neutralizing antibodies. Compared to controls, vaccinees had significantly lower peak viral RNA loads, and one vaccine recipient remained completely virus-free, even in lymphoid tissues. There was a trend for the protein-only vaccine to yield better protection than the combined modality approach. CONCLUSION: Protein-only immunogens induced significant protection against heterologous viruses encoding env from locally circulating viruses.


Asunto(s)
Proteínas gp160 de Envoltorio del VIH/inmunología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Administración Oral , Animales , Anticuerpos Antivirales/inmunología , ADN Viral/inmunología , Modelos Animales de Enfermedad , Esquema de Medicación , Productos del Gen gag/inmunología , Productos del Gen tat/inmunología , Proteínas gp160 de Envoltorio del VIH/genética , Macaca mulatta , ARN Viral/inmunología , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Virus de la Inmunodeficiencia de los Simios/genética , Vacunación/métodos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Carga Viral , Proteínas Virales/inmunología
12.
J Interferon Cytokine Res ; 26(6): 380-9, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16734558

RESUMEN

Controlled release of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein by albumin-heparin microparticles administered via intramuscular vaccination in conjunction with HIV DNA vaccines stimulated HIV Gag-specific immune responses. In the murine model, Gag-specific cytotoxic T lymphocyte (CTL) and T helper (Th) responses were significantly enhanced by administration of murine GM-CSF microparticles. This effect was comparable to a GM-CSF encoded plasmid. In three of four rhesus monkeys, enhancement of Gag-specific antibody (Ab), Th, and CTL responses was observed 1 month after the first immunization with coadministration of human GM-CSF microparticles and HIV Gag plasmid. The second, third, and fourth booster immunizations, however, did not increase the Gag-specific immune responses. Subsequent application of Gag protein in complete Freund's adjuvant (CFA) significantly enhanced Ab and Th, but not CTL. However, Gag-specific CTL response was triggered by cytokine and Gag p55-encapsulated microparticles in all animals. The strategy of priming immune responses by coadministration of cytokine microparticles and DNA vaccines, followed by boosting with cytokine and antigen protein-encapsulated microparticles, may prove effective in improving an HIV DNA vaccine design.


Asunto(s)
Productos del Gen gag/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , VIH , Vacunas de ADN/genética , Albúminas/farmacocinética , Albúminas/ultraestructura , Animales , Pruebas Inmunológicas de Citotoxicidad , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Heparina/farmacocinética , Heparina/ultraestructura , Humanos , Inyecciones Intramusculares , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Microesferas , Modelos Animales , Músculo Esquelético/inmunología , Plásmidos , Proteínas Recombinantes , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología
13.
J Acquir Immune Defic Syndr ; 73(4): 365-373, 2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27792681

RESUMEN

BACKGROUND: Preexposure prophylaxis using antiretroviral agents has been shown to effectively prevent human immunodeficiency virus type 1 (HIV-1) acquisition in high-risk populations. However, the efficacy of these regimens is highly variable, which is thought to be largely due to the varying degrees of adherence to a daily intervention in the populations. Passive immunization using broadly neutralizing antibodies (bNAbs) against HIV-1, with their relatively long half-life and favorable safety profile, could provide an alternative to daily preexposure prophylaxis. However, most bNAbs have a limited breadth, only neutralizing 70%-90% of all HIV-1 strains. METHODS: To overcome the problem of limited antiviral breadth, we proposed that targeting human CD4 and HIV-1 envelope proteins simultaneously may improve virus-neutralization breadth and potency. Therefore, we constructed bispecific antibodies (biAbs) using single-chain variable fragments of anti-gp120 bNAbs fused to ibalizumab (iMab), a humanized monoclonal antibody that binds human CD4, the primary receptor for HIV-1. RESULTS: Some of our biAbs neutralized 100% of HIV-1 strains tested in vitro at clinically achievable concentrations. Distinct neutralization patterns were observed in this panel of biAbs. Those biAbs with specificity for the CD4-binding site on gp120 demonstrated 100% breadth, as well as slightly improved potency compared with iMab. In contrast, biAbs with specificity for the V1-V2 apex epitope or the V3-glycan epitope on gp120 demonstrated dramatically improved potency; some showed limited gain in neutralization breadth, whereas others (eg, PGT128-LM52 and 123-iMab) improved to 100% breadth. CONCLUSION: Our data suggest that this panel of iMab-based biAbs could be used to probe the parameters for potent HIV-1 neutralization. Moreover, a few of these biAbs warrant further studies and possibly clinical development.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Anticuerpos Neutralizantes/inmunología , Citocinas/metabolismo , Células HeLa , Humanos , Pruebas de Neutralización
14.
mBio ; 6(2)2015 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-25900654

RESUMEN

UNLABELLED: Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Ig(mim2), CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4(+) T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection. IMPORTANCE: SIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4(+) T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Reacciones Cruzadas , Anticuerpos Anti-VIH/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Gorilla gorilla , Humanos , Concentración 50 Inhibidora , Pruebas de Neutralización , Pan troglodytes , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación
15.
Nat Biotechnol ; 31(11): 1047-52, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24097413

RESUMEN

Ibalizumab is a humanized monoclonal antibody that binds human CD4--a key receptor for HIV--and blocks HIV-1 infection. However, HIV-1 strains with mutations resulting in loss of an N-linked glycan from the V5 loop of the envelope glycoprotein gp120 are resistant to ibalizumab. Previous structural analysis suggests that this glycan fills a void between the gp120 V5 loop and the ibalizumab light chain, perhaps causing steric hindrance that disrupts viral entry. If this void contributes to HIV-1 resistance to ibalizumab, we reasoned that 'refilling' it by engineering an N-linked glycan into the ibalizumab light chain at a position spatially proximal to gp120 V5 may restore susceptibility to ibalizumab. Indeed, one such ibalizumab variant neutralized 100% of 118 diverse HIV-1 strains tested in vitro, including 10 strains resistant to parental ibalizumab. These findings demonstrate that the strategic placement of a glycan in the variable region of a monoclonal antibody can substantially enhance its activity.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/farmacología , Antígenos CD4/metabolismo , Reacciones Cruzadas , Farmacorresistencia Viral/efectos de los fármacos , Células HEK293 , Inhibidores de Fusión de VIH/química , Inhibidores de Fusión de VIH/farmacología , VIH-1/aislamiento & purificación , Humanos , Modelos Moleculares , Polisacáridos/química , Polisacáridos/farmacología , Relación Estructura-Actividad
16.
PLoS One ; 5(7): e11689, 2010 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-20657739

RESUMEN

BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.


Asunto(s)
Vacunas contra el SIDA/inmunología , Genes env/genética , VIH-1/inmunología , VIH-1/patogenicidad , Vacunas contra el SIDA/genética , Animales , Anticuerpos Neutralizantes/inmunología , Modelos Animales de Enfermedad , Evolución Molecular , VIH-1/genética , Macaca mulatta , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa
17.
DNA Cell Biol ; 28(3): 103-8, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19182996

RESUMEN

We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFP-positive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinase-deficient (Tk(-)) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk(-) rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins.


Asunto(s)
Genes env , Proteínas Fluorescentes Verdes/genética , VIH/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Virus Vaccinia/genética , Línea Celular , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , VIH/metabolismo , Humanos , Proteínas Recombinantes/metabolismo , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Virus Vaccinia/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo
18.
PLoS Negl Trop Dis ; 2(7): e265, 2008 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-18648516

RESUMEN

BACKGROUND: Individuals living in sub-Saharan Africa represent 10% of the world's population but almost 2/3 of all HIV-1/AIDS cases. The disproportionate HIV-1 infection rates in this region may be linked to helminthic parasite infections that affect many individuals in the developing world. However, the hypothesis that parasite infection increases an individual's susceptibility to HIV-1 has never been prospectively tested in a relevant in vivo model. METHODOLOGY/PRINCIPAL FINDINGS: We measured whether pre-existing infection of rhesus monkeys with a parasitic worm would facilitate systemic infection after mucosal AIDS virus exposure. Two groups of animals, one consisting of normal monkeys and the other harboring Schistosoma mansoni, were challenged intrarectally with decreasing doses of R5-tropic clade C simian-human immunodeficiency virus (SHIV-C). Systemic infection occurred in parasitized monkeys at viral doses that remained sub-infectious in normal hosts. In fact, the 50% animal infectious (AID(50)) SHIV-C dose was 17-fold lower in parasitized animals compared to controls (P<0.001). Coinfected animals also had significantly higher peak viral RNA loads than controls (P<0.001), as well as increased viral replication in CD4(+) central memory cells (P = 0.03). CONCLUSIONS/SIGNIFICANCE: Our data provide the first direct evidence that acute schistosomiasis significantly increases the risk of de novo AIDS virus acquisition, and the magnitude of the effect suggests that control of helminth infections may be a useful public health intervention to help decrease the spread of HIV-1.


Asunto(s)
Susceptibilidad a Enfermedades , Infecciones por VIH/inmunología , Macaca mulatta , Esquistosomiasis mansoni/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Modelos Animales de Enfermedad , Femenino , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Macaca mulatta/parasitología , Macaca mulatta/virología , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Factores de Riesgo , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/parasitología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/clasificación , Carga Viral , Replicación Viral
19.
Mol Ther ; 15(5): 1007-15, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17356539

RESUMEN

Transfection of DNA vaccines with chemokines may recruit dendritic cells (DCs) locally to capture the antigenic genes and their gene products to generate enhanced CD8(+) cytotoxic T lymphocytes (CTLs). In this study, we investigated the effects of macrophage inflammatory protein (MIP)-1 alpha, MIP-3 alpha, and MIP-3beta on human immunodeficiency virus (HIV) Gag DNA vaccination. The chemokine plasmids markedly enhanced the local infiltration of inflammatory cells and increased the presence of CD11c(+) B7.2(+)-activated DCs. MIP-1 alpha and MIP-3 alpha were potent adjuvants in augmenting CTLs and afforded strong protection to immunized animals against challenge with vaccinia virus expressing Gag (vv-Gag). However, decreased humoral response was observed. MIP-3beta plasmid did not dramatically alter immunity. The chemokine inoculation time with respect to DNA vaccine priming was also investigated. The injection of pMIP-3 alpha three days before Gag plasmid (pGag) vaccination markedly increased specific CTLs compared with simultaneous injection and led to higher protection against vv-Gag. Immunity was also shifted toward a T-helper type-1 (Th1) response. In contrast, inoculation with pMIP-3 alpha three days after pGag vaccination shifted immunity toward a Th2 response. Our data suggest that administration of a chemokine with DNA vaccines offers a valuable strategy to modulate the efficacy and polarization of specific immunity and that chemokine-antigen timing is critical in determining overall biological effects.


Asunto(s)
Productos del Gen gag/inmunología , VIH/inmunología , Proteínas Inflamatorias de Macrófagos/inmunología , Vacunas de ADN/inmunología , Animales , Línea Celular , Quimiocina CCL4 , Quimiotaxis/inmunología , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Productos del Gen gag/genética , VIH/genética , Humanos , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Plásmidos/administración & dosificación , Plásmidos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Linfocitos T Citotóxicos/inmunología , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética
20.
Infect Immun ; 75(4): 1751-6, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17283092

RESUMEN

We tested the hypothesis that helminth parasite coinfection would intensify viremia and accelerate disease progression in monkeys chronically infected with an R5 simian-human immunodeficiency virus (SHIV) encoding a human immunodeficiency virus type 1 (HIV-1) clade C envelope. Fifteen rhesus monkeys with stable SHIV-1157ip infection were enrolled into a prospective, randomized trial. These seropositive animals had undetectable viral RNA and no signs of immunodeficiency. Seven animals served as virus-only controls; eight animals were exposed to Schistosoma mansoni cercariae. From week 5 after parasite exposure onward, coinfected animals shed eggs in their feces, developed eosinophilia, and had significantly higher mRNA expression of the T-helper type 2 cytokine interleukin-4 (P = 0.001) than animals without schistosomiasis. Compared to virus-only controls, viral replication was significantly increased in coinfected monkeys (P = 0.012), and the percentage of their CD4(+) CD29(+) memory cells decreased over time (P = 0.05). Thus, S. mansoni coinfection significantly increased viral replication and induced T-cell subset alterations in monkeys with chronic SHIV clade C infection.


Asunto(s)
Esquistosomiasis mansoni/complicaciones , Síndrome de Inmunodeficiencia Adquirida del Simio/complicaciones , Virus de la Inmunodeficiencia de los Simios/fisiología , Viremia , Animales , Antígenos CD4/análisis , Modelos Animales de Enfermedad , Eosinofilia , Heces/parasitología , Integrina beta1/análisis , Interleucina-4/biosíntesis , Subgrupos Linfocitarios/inmunología , Macaca mulatta , ARN Viral/sangre , Schistosoma mansoni , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Carga Viral , Replicación Viral
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