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1.
Cell ; 178(3): 567-584.e19, 2019 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-31348886

RESUMEN

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/clasificación , Linfocitos B/citología , Linfocitos B/metabolismo , Cristalografía por Rayos X , Femenino , Células HEK293 , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/clasificación , VIH-1/metabolismo , Humanos , Macaca mulatta , Masculino , Péptidos/química , Estructura Terciaria de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-34551978

RESUMEN

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Disulfuros/química , Epítopos/inmunología , Glicoproteínas/inmunología , Metapneumovirus/inmunología , Mutación , Animales , Glicoproteínas/genética , Humanos , Inmunización , Macaca , Metapneumovirus/genética , Ratones , Regiones Promotoras Genéticas
3.
Am J Primatol ; 85(11): e23554, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37771291

RESUMEN

Demand for nonhuman primates in research has increased over the past several years, while nonhuman primate supply remains a challenge in the United States. Global nonhuman primate supply issues make it increasingly important to maximize domestic colony production. To explore how housing conditions across primate breeding colonies impact infant survival and animal production more broadly, we collected medical records from 7959 rhesus macaques (Macaca mulatta) and 492 pigtail macaques (Macaca nemestrina) across seven breeding facilities and used generalized mixed-effect modeling to determine prenatal and infant survival odds by housing type and group size. Infant survival odds for each housing type and group size varied for prenatal, neonatal, early infant, and late infant age groups. Odds of prenatal survival were lowest in paired indoor housing and small and medium outdoor groups. No housing type performed better than large outdoor groups for neonatal survival. Odds of early infant survival was greatest in indoor and mixed indoor/outdoor housing compared to large outdoor enclosures. Large outdoor housing was associated with higher survival odds for late infant survival compared to small and medium outdoor housing. These results may influence housing choices at macaque breeding facilities hoping to maximize infant success, although there are relative care costs, the promotion of species-typical behaviors, and infrastructure factors to also consider. Our study used an interinstitutional collaboration that allowed for the analysis of more infant macaque medical records than ever before and used the broad variations across the seven national primate research centers to make the results applicable to many other facilities housing macaques.


Asunto(s)
Cruzamiento , Vivienda para Animales , Humanos , Embarazo , Femenino , Animales , Macaca mulatta , Macaca nemestrina
4.
Proc Natl Acad Sci U S A ; 115(48): 12265-12270, 2018 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-30420505

RESUMEN

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.


Asunto(s)
Virus de la Parainfluenza 1 Humana/inmunología , Virus de la Parainfluenza 2 Humana/inmunología , Virus de la Parainfluenza 3 Humana/inmunología , Virus de la Parainfluenza 4 Humana/inmunología , Infecciones por Respirovirus/inmunología , Proteínas Virales de Fusión/química , Vacunas Virales/química , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Microscopía por Crioelectrón , Femenino , Humanos , Macaca mulatta , Masculino , Ratones , Virus de la Parainfluenza 1 Humana/química , Virus de la Parainfluenza 1 Humana/genética , Virus de la Parainfluenza 2 Humana/química , Virus de la Parainfluenza 2 Humana/genética , Virus de la Parainfluenza 3 Humana/química , Virus de la Parainfluenza 3 Humana/genética , Virus de la Parainfluenza 4 Humana/química , Virus de la Parainfluenza 4 Humana/genética , Infecciones por Virus Sincitial Respiratorio , Infecciones por Respirovirus/prevención & control , Infecciones por Respirovirus/virología , Proteínas Virales de Fusión/administración & dosificación , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/inmunología
6.
J Immunol ; 193(10): 5088-98, 2014 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-25305312

RESUMEN

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. Our data previously demonstrated that A. phagocytophilum induces an immunopathologic response by activating IFN-γ production through the Stat1 signaling pathway. In this study, we investigated the broader role of Stat1 signaling in the host response to infection with A. phagocytophilum. In Stat1 knockout (KO) compared with wild-type mice, A. phagocytophilum infection was more highly pathogenic as characterized by the unanticipated development of clinical signs in mice including markedly increased splenomegaly, more severe inflammatory splenic and hepatic histopathology, >100-fold higher blood and splenic bacterial loads, and more elevated proinflammatory cytokine/chemokine responses in serum. CD4(+) and CD8(+) T lymphocyte populations were significantly expanded in spleens of A. phagocytophilum-infected Stat1 KO mice compared with wild-type mice. The leukocyte infiltrates in the livers and spleens of A. phagocytophilum-infected Stat1 KO mice also contained expansions in neutrophil and monocyte/macrophage populations. Importantly, A. phagocytophilum-infected Stat1 KO mice did not demonstrate induction of inducible NO synthase in splenocytes. These results show that Stat1 plays an important role in controlling bacterial loads but also by unexpectedly providing an undefined mechanism for dampening of the immunopathologic response observed with A. phagocytophilum infection.


Asunto(s)
Anaplasma phagocytophilum/inmunología , Anaplasmosis/inmunología , Hígado/inmunología , Factor de Transcripción STAT1/inmunología , Bazo/inmunología , Anaplasmosis/genética , Anaplasmosis/microbiología , Anaplasmosis/patología , Animales , Carga Bacteriana , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/patología , Expresión Génica , Inmunomodulación , Hígado/microbiología , Hígado/patología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Noqueados , Monocitos/inmunología , Monocitos/microbiología , Monocitos/patología , Neutrófilos/inmunología , Neutrófilos/microbiología , Neutrófilos/patología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/genética , Índice de Severidad de la Enfermedad , Transducción de Señal , Bazo/microbiología , Bazo/patología
7.
Infect Immun ; 82(2): 818-29, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24478095

RESUMEN

Malaria transmission-blocking vaccines (TBVs) represent a promising approach for the elimination and eradication of this disease. AnAPN1 is a lead TBV candidate that targets a surface antigen on the midgut of the obligate vector of the Plasmodium parasite, the Anopheles mosquito. In this study, we demonstrated that antibodies targeting AnAPN1 block transmission of Plasmodium falciparum and Plasmodium vivax across distantly related anopheline species in countries to which malaria is endemic. Using a biochemical and immunological approach, we determined that the mechanism of action for this phenomenon stems from antibody recognition of a single protective epitope on AnAPN1, which we found to be immunogenic in murine and nonhuman primate models and highly conserved among anophelines. These data indicate that AnAPN1 meets the established target product profile for TBVs and suggest a potential key role for an AnAPN1-based panmalaria TBV in the effort to eradicate malaria.


Asunto(s)
Anopheles/parasitología , Transmisión de Enfermedad Infecciosa/prevención & control , Proteínas de Insectos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Malaria Vivax/prevención & control , Animales , Femenino , Proteínas de Insectos/administración & dosificación , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/transmisión , Malaria Vivax/transmisión , Masculino , Ratones , Ratones Endogámicos BALB C
8.
PLoS Negl Trop Dis ; 18(2): e0011993, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38408129

RESUMEN

Spotted fever group rickettsiae are tick-borne obligate intracellular bacteria that infect microvascular endothelial cells. Humans and mammalian infection results in endothelial cell barrier dysfunction and increased vascular permeability. We previously demonstrated that treatment of Rickettsia parkeri-infected cells with the calcium channel blocker benidipine significantly delayed vascular barrier permeability. Thus, we hypothesized that benidipine, known to be safe and effective for other clinical processes, could reduce rickettsia-induced vascular permeability in vivo in an animal model of spotted fever rickettsiosis. Based on liver, lung and brain vascular FITC-dextran extravasation studies, benidipine did not reliably impact vascular permeability. However, it precipitated a deleterious effect on responses to control sublethal R. parkeri infection. Animals treated with benidipine alone had no clinical signs or changes in histopathology and splenic immune cell distributions. Benidipine-treated infected animals had marked increases in tissue and blood bacterial loads, more extensive inflammatory histopathologic injury, and changes in splenic architecture and immune cell distributions potentially reflecting diminished Ca2+ signaling, reduced innate immune cell activation, and loss of rickettsial propagation control. Impaired T cell activation by R. parkeri antigen in the presence of benidipine was confirmed in vitro with the use of NKT cell hybridomas. The unexpected findings stand in stark contrast to recent discussions of the benefits of calcium channel blockers for viral infections and chronic infectious or inflammatory diseases. A role for calcium channel blockers in exacerbation of human rickettsiosis and acute inflammatory infections should be evaluated by a retrospective review of patient's outcomes and medications.


Asunto(s)
Dihidropiridinas , Infecciones por Rickettsia , Rickettsia , Rickettsiosis Exantemáticas , Humanos , Ratones , Animales , Modelos Animales de Enfermedad , Bloqueadores de los Canales de Calcio , Células Endoteliales/patología , Infecciones por Rickettsia/microbiología , Rickettsia/fisiología , Rickettsiosis Exantemáticas/patología , Inmunidad Innata , Mamíferos
9.
Front Cell Infect Microbiol ; 13: 1187267, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37228668

RESUMEN

Rickettsial agents are a diverse group of alpha-proteobacteria within the order Rickettsiales, which possesses two families with human pathogens, Rickettsiaceae and Anaplasmataceae. These obligate intracellular bacteria are most frequently transmitted by arthropod vectors, a first step in the pathogens' avoidance of host cell defenses. Considerable study of the immune responses to infection and those that result in protective immunity have been conducted. Less study has focused on the initial events and mechanism by which these bacteria avoid the innate immune responses of the hosts to survive within and propagate from host cells. By evaluating the major mechanisms of evading innate immunity, a range of similarities among these bacteria become apparent, including mechanisms to escape initial destruction in phagolysosomes of professional phagocytes, those that dampen the responses of innate immune cells or subvert signaling and recognition pathways related to apoptosis, autophagy, proinflammatory responses, and mechanisms by which these microbes attach to and enter cells or those molecules that trigger the host responses. To illustrate these principles, this review will focus on two common rickettsial agents that occur globally, Rickettsia species and Anaplasma phagocytophilum.


Asunto(s)
Anaplasma phagocytophilum , Infecciones por Rickettsia , Rickettsia , Humanos , Infecciones por Rickettsia/microbiología , Inmunidad Innata , Autofagia
10.
Vaccine ; 41(10): 1735-1742, 2023 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-36764908

RESUMEN

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of ≥ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN.


Asunto(s)
Virus BK , Enfermedades Renales , Infecciones por Polyomavirus , Poliomavirus , Infecciones Tumorales por Virus , Vacunas de Partículas Similares a Virus , Animales , Humanos , Macaca mulatta , Viremia/prevención & control , Anticuerpos Neutralizantes
11.
Cell Rep ; 32(5): 107981, 2020 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-32755575

RESUMEN

The HIV fusion peptide (FP) is a promising vaccine target. FP-directed monoclonal antibodies from vaccinated macaques have been identified that neutralize up to ∼60% of HIV strains; these vaccinations, however, have involved ∼1 year with an extended neutralization-eclipse phase without measurable serum neutralization. Here, in 32 macaques, we test seven vaccination regimens, each comprising multiple immunizations of FP-carrier conjugates and HIV envelope (Env) trimers. Comparisons of vaccine regimens reveal FP-carrier conjugates to imprint cross-clade neutralizing responses and a cocktail of FP conjugate and Env trimer to elicit the earliest broad responses. We identify a signature, appearing as early as week 6 and involving the frequency of B cells recognizing both FP and Env trimer, predictive of vaccine-elicited breadth ∼1 year later. Immune monitoring of B cells in response to vaccination can thus enable vaccine insights even in the absence of serum neutralization, here identifying FP imprinting, cocktail approach, and early signature as means to improve FP-directed vaccine responses.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , Monitorización Inmunológica , Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas contra el SIDA/inmunología , Animales , Antígenos VIH/inmunología , Infecciones por VIH/inmunología , Hemocianinas/metabolismo , Inmunización , Macaca mulatta , Masculino , Polisacáridos/metabolismo , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología
12.
Comp Med ; 59(1): 78-82, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19295057

RESUMEN

The causes and treatments of pancreatitis have been studied in diverse species, but the canine pancreatitis model has been used most often due to its similarities to the condition in humans. Although pancreatitis in dogs can be induced readily by numerous methods, managing these dogs can be difficult because they often develop severe abdominal pain, vomiting, inappetance, and lethargy. In an effort to study pancreatitis, we performed a pilot study to determine whether an endoscopic pancreatic procedure would be possible in a dog and whether, through various manipulations, a new method of inducing pancreatitis could be developed. The model uses endoscopic retrograde cholangiopancreatography (ERCP), a common procedure in human gastroenterology that has been associated with postprocedural pancreatitis. Although all 8 dogs used in developing the ERCP model had both biochemical and histologic changes consistent with pancreatitis, 7 of the 8 dogs remained free of classic clinical signs of the disease. This method is presented as a refinement of a canine model and presents an alternative method of inducing pancreatitis, with decreased risk of developing associated clinical signs.


Asunto(s)
Colangiopancreatografia Retrógrada Endoscópica/efectos adversos , Colangiopancreatografia Retrógrada Endoscópica/métodos , Páncreas/patología , Pancreatitis/etiología , Pancreatitis/patología , Enfermedad Aguda , Amilasas/sangre , Animales , Modelos Animales de Enfermedad , Perros , Fluoroscopía , Lipasa/sangre , Masculino , Pancreatitis/sangre , Proyectos Piloto , Complicaciones Posoperatorias
13.
Res Vet Sci ; 127: 76-81, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31678456

RESUMEN

Antimicrobial resistance is increasing within the porcine industry with consequential high impact on human health, leading to a need for new antimicrobials. Lately, the scientific community has turned its interest towards natural compounds, and different essential oils have been tested on spermatozoa for preliminary assessment of toxicity before considering them as good substitutes for standard antibiotics. The aim of the present work was to investigate the potential antimicrobial effect of Melaleuca alternifolia and Rosmarinus officinalis essential oils, already evaluated for toxicity, on swine artificial insemination doses deprived of spermatozoa and stored at 16 °C for 5 days. This was accomplished by setting up an in vitro model with a standardized quantity of E. coli. Essential oils, previously chemo-characterized by means of gas chromatography, were tested at 0.2 and 0.4 mg/ml. Analyses, performed at 24 and 120 h, included optical density evaluation, bacterial DNA quantification by qPCR, and colony count. The results demonstrate that both Melaleuca alternifolia and Rosmarinus officinalis essential oils, at a concentration of 0.4 mg/ml, are capable of delivering similar effects to ampicillin, used as control, on the experimental samples. At the lower concentration, M. alternifolia essential oil seemed more effective when compared to R. officinalis. Overall, these findings strengthen the hypothesis of the potential use of phyto-complexes as antimicrobial agents for reproductive biotechnologies.


Asunto(s)
Antiinfecciosos/farmacología , Escherichia coli/efectos de los fármacos , Aceites Volátiles/farmacología , Semen/efectos de los fármacos , Aceite de Árbol de Té/farmacología , Animales , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Semen/microbiología , Sus scrofa
14.
PLoS One ; 14(4): e0215163, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30995238

RESUMEN

The vaccine elicitation of broadly neutralizing responses is a central goal of HIV research. Recently, we elicited cross-clade neutralizing responses against the N terminus of the fusion peptide (FP), a critical component of the HIV-entry machinery. While the consistency of the elicited cross-clade neutralizing responses was good in mice, it was poor in guinea pigs: after seven immunizations comprising either envelope (Env) trimer or FP coupled to a carrier, serum from only one of five animals could neutralize a majority of a cross-clade panel of 19 wild-type strains. Such a low response rate-only 20%-made increasing consistency an imperative. Here, we show that additional Env-trimer immunizations could boost broad FP-directed neutralizing responses in a majority of immunized animals. The first boost involved a heterologous Env trimer developed from the transmitted founder clade C strain of donor CH505, and the second boost involved a cocktail that combined the CH505 trimer with a trimer from the BG505 strain. After boosting, sera from three of five animals neutralized a majority of the 19-strain panel and serum from a fourth animal neutralized 8 strains. We demonstrate that cross-reactive serum neutralization targeted the FP by blocking neutralization with soluble fusion peptide. The FP competition revealed two categories of elicited responses: an autologous response to the BG505 strain of high potency (~10,000 ID50), which was not competed by soluble FP, and a heterologous response of lower potency, which was competed by soluble FP. While the autologous response could increase rapidly in response to Env-trimer boost, the heterologous neutralizing response increased more slowly. Overall, repetitive Env-trimer immunizations appeared to boost low titer FP-carrier primed responses to detectable levels, yielding cross-clade neutralization. The consistent trimer-boosted neutralizing responses described here add to accumulating evidence for the vaccine utility of the FP site of HIV vulnerability.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunización Secundaria , Péptidos/farmacocinética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/farmacocinética , Animales , Cobayas , Infecciones por VIH/prevención & control , VIH-1/genética , Péptidos/genética , Péptidos/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/farmacología
15.
Comp Med ; 58(6): 568-79, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19149414

RESUMEN

Porcine models have become increasingly popular in cardiovascular research. The standard farm pig rapidly increases in body weight and size, potentially confounding serial measurements of cardiac function and morphology. We developed an adult porcine model that does not show physiologic increases in heart mass during the study period and is suitable for long-term study. We compared adult minipigs with the commonly used adolescent Yorkshire swine. Myocardial infarction was induced in adult Göttingen minipigs and adolescent Yorkshire swine by occlusion of the left anterior descending coronary artery followed by reperfusion. At 8 wk after infarction, the left ventricular ejection fraction was 34.1 +/- 2.3% in minipigs and 30.7 +/- 2.0% in Yorkshire swine. The left ventricular end-diastolic mass in Yorkshire pigs assessed by magnetic resonance imaging increased 17 +/- 5 g, from 42.6 +/- 4.3 g at week 1 after infarction to 52.8 +/- 6.6 g at week 8, whereas it remained unchanged in minipigs. Cardiac anatomy and physiology in adult minipigs were evaluated invasively by angiography and noninvasively by Multidetector Computed Tomography and by Magnetic Resonance Imaging at 1.5 T and 3 T prior to myocardial infarction and during folow-up. This porcine heart failure model is reproducible, mimics the pathophysiology in patients who have experienced myocardial infarction, and is suitable for imaging studies. New heart failure therapies and devices can be tested preclinically in this adult animal model of chronic heart failure.


Asunto(s)
Insuficiencia Cardíaca/etiología , Infarto del Miocardio/complicaciones , Animales , Modelos Animales de Enfermedad , Electrocardiografía , Femenino , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/terapia , Angiografía por Resonancia Magnética , Especificidad de la Especie , Sus scrofa , Porcinos , Porcinos Enanos , Tomografía Computarizada por Rayos X , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/fisiopatología
16.
Front Immunol ; 9: 710, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29686681

RESUMEN

Human granulocytic anaplasmosis, caused by the tick-transmitted Anaplasma phagocytophilum, is not controlled by innate immunity, and induces a proinflammatory disease state with innate immune cell activation. In A. phagocytophilum murine infection models, hepatic injury occurs with production of IFNγ thought to be derived from NK, NKT cells, and CD8 T lymphocytes. Specific A. phagocytophilum ligands that drive inflammation and disease are not known, but suggest a clinical and pathophysiologic basis strikingly like macrophage activation syndrome (MAS) and hemophagocytic syndrome (HPS). We studied in vivo responses of NK, NKT, and CD8 T lymphocytes from infected animals for correlates of lymphocyte-mediated cytotoxicity and examined in vitro interactions with A. phagocytophilum-loaded antigen-presenting cells (APCs). Murine splenocytes were examined and found deficient in cytotoxicity as determined by CD107a expression in vitro for specific CTL effector subsets as determined by flow cytometry. Moreover, A. phagocytophilum-loaded APCs did not lead to IFNγ production among CTLs in vitro. These findings support the concept of impaired cytotoxicity with A. phagocytophilum presentation by APCs that express MHC class I and that interact with innate and adaptive immune cells with or after infection. The findings strengthen the concept of an enhanced proinflammatory phenotype, such as MAS and HPS disease states as the basis of disease and severity with A. phagocytophilum infection, and perhaps by other obligate intracellular bacteria.


Asunto(s)
Anaplasma phagocytophilum/inmunología , Citotoxicidad Inmunológica , Interacciones Huésped-Patógeno/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Ehrlichiosis/inmunología , Ehrlichiosis/microbiología , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/inmunología , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo
17.
Cell Rep ; 22(7): 1798-1809, 2018 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-29444432

RESUMEN

Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ∼10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Membrana Celular/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Semivida , Humanos , Pruebas de Neutralización , Polisacáridos/metabolismo , Unión Proteica
18.
Nat Med ; 24(6): 857-867, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29867235

RESUMEN

A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Secuencia de Aminoácidos , Animales , Femenino , Cobayas , VIH-1/efectos de los fármacos , Inmunización , Macaca mulatta , Ratones Endogámicos C57BL , Modelos Moleculares , Pruebas de Neutralización , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
19.
FEMS Immunol Med Microbiol ; 49(3): 374-86, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17286796

RESUMEN

Msp2 is Anaplasma phagocytophilum's immunodominant protein. Antigenic variability with msp2 gene conversion may drive differential immunopathology with infection by bacteria of different in vitro passage intervals. We examined msp2 transcript variation and its relationship to histopathology, T-cell and antibody responses in mice infected with differentially passaged A. phagocytophilum. Hepatic inflammation peaked on day 2-4 with low passage bacteria and on day 4-7 with high passage bacteria infection. Nineteen msp2 variant transcripts were identified. The low and high passage inocula shared four, but differed in one and two msp2 transcript variants, respectively. After infection, three and two msp2 variants were only identified in low or high passage infected mice. However, per mouse, msp2 variant profiles were unique with no evident expression program. In low and high passage bacteria-infected mice, splenocytes proliferated to whole A. phagocytophilum at day 7-10, diminishing thereafter. Weak mitogenic responses to whole bacteria were detected in mock and infected mice at d0 and sporadically thereafter. Essentially no lymphoproliferation or IFN-gamma production resulted from stimulation by six Msp2 hypervariable region proteins, although antibodies were detected to all, including cross-reactions. Differential A. phagocytophilum Msp2 expression is unrelated to T-cell response and unlikely to induce the cellular immunopathology underlying disease manifestations.


Asunto(s)
Anaplasma phagocytophilum/inmunología , Variación Antigénica , Proteínas de la Membrana Bacteriana Externa/inmunología , Ehrlichiosis/microbiología , Interferón gamma/biosíntesis , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/genética , Proliferación Celular , Modelos Animales de Enfermedad , Ehrlichiosis/inmunología , Ehrlichiosis/patología , Histocitoquímica , Hígado/microbiología , Hígado/patología , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Bazo/inmunología , Transcripción Genética
20.
Cell Rep ; 19(4): 719-732, 2017 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-28445724

RESUMEN

While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralization character). To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID50) proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , VIH-1/metabolismo , Polisacáridos/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Especificidad de Anticuerpos , Sitios de Unión , Antígenos CD4/química , Antígenos CD4/metabolismo , Cristalografía por Rayos X , Epítopos/inmunología , Glicosilación , Cobayas , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , Humanos , Inmunización , Macaca mulatta , Simulación de Dinámica Molecular , Polisacáridos/deficiencia , Polisacáridos/metabolismo , Estructura Cuaternaria de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
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