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1.
J Biol Chem ; 289(11): 7630-40, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24497632

RESUMO

A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.


Assuntos
Arginina/química , Proteína gp41 do Envelope de HIV/química , HIV-1/química , Peptídeos/química , Motivos de Aminoácidos , Fusão Celular , Separação Celular , Clonagem Molecular , Biologia Computacional , Citometria de Fluxo , Células HEK293 , HIV-1/fisiologia , Humanos , Cinética , Lisina/química , Modelos Moleculares , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Replicação Viral
2.
Antimicrob Agents Chemother ; 59(2): 1329-33, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25421473

RESUMO

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Rifampina/farmacologia
3.
Biophys J ; 105(3): 657-66, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23931314

RESUMO

Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (-1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.


Assuntos
Membrana Celular/química , Proteína gp41 do Envelope de HIV/química , Fragmentos de Peptídeos/química , Animais , Membrana Celular/metabolismo , Colesterol/metabolismo , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/química , Humanos , Lipoilação , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Linfócitos T/metabolismo
4.
Antimicrob Agents Chemother ; 57(6): 2511-21, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23507278

RESUMO

The emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against both P. aeruginosa and S. aureus under challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Arginina/química , Dicroísmo Circular , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Triptofano/química
5.
J Gen Virol ; 94(Pt 1): 1-19, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23079381

RESUMO

The human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) pandemic is amongst the most important current worldwide public health threats. While much research has been focused on AIDS vaccines that target the surface viral envelope (Env) protein, including gp120 and the gp41 ectodomain, the C-terminal tail (CTT) of gp41 has received relatively little attention. Despite early studies highlighting the immunogenicity of a particular CTT sequence, the CTT has been classically portrayed as a type I membrane protein limited to functioning in Env trafficking and virion incorporation. Recent studies demonstrate, however, that the Env CTT has other important functions. The CTT has been shown to additionally modulate Env ectodomain structure on the cell and virion surface, affect Env reactivity and viral sensitivity to conformation-dependent neutralizing antibodies, and alter cell-cell and virus-cell fusogenicity of Env. This review provides an overview of the Env structure and function with a particular emphasis on the CTT and recent studies that highlight its functionally rich nature.


Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , HIV/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Animais , HIV/química , HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Relação Estrutura-Atividade , Proteínas do Envelope Viral/imunologia
6.
J Biol Chem ; 286(31): 27156-66, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21659530

RESUMO

Although the HIV-1 Env gp120 and gp41 ectodomain have been extensively characterized in terms of structure and function, similar characterizations of the C-terminal tail (CTT) of HIV gp41 remain relatively limited and contradictory. The current study was designed to examine in detail CTT sequence conservation relative to gp120 and the gp41 ectodomain and to examine the conservation of predicted physicochemical and structural properties across a number of divergent HIV clades and groups. Results demonstrate that CTT sequences display intermediate levels of sequence evolution and diversity in comparison to the more diverse gp120 and the more conserved gp41 ectodomain. Despite the relatively high level of CTT sequence variation, the physicochemical properties of the lentivirus lytic peptide domains (LLPs) within the CTT are evidently highly conserved across clades/groups. Additionally, predictions using PEP-FOLD indicate a high level of structural similarity in the LLP regions that was confirmed by circular dichroism measurements of secondary structure of LLP peptides from clades B, C, and group O. Results demonstrate that LLP peptides adopt helical structure in the presence of SDS or trifluoroethanol but are predominantly unstructured in aqueous buffer. Thus, these data for the first time demonstrate strong conservations of characteristic CTT physicochemical and structural properties despite substantial sequence diversity, apparently indicating a delicate balance between evolutionary pressures and the conservation of CTT structure and associated functional roles in virus replication.


Assuntos
Sequência Conservada , Proteína gp41 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Replicação Viral/fisiologia , Sequência de Aminoácidos , Dicroísmo Circular , Proteína gp41 do Envelope de HIV/química , HIV-1/classificação , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
7.
PLoS One ; 17(9): e0274815, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36112657

RESUMO

The absence of novel antibiotics for drug-resistant and biofilm-associated infections is a global public health crisis. Antimicrobial peptides explored to address this need have encountered significant development challenges associated with size, toxicity, safety profile, and pharmacokinetics. We designed PLG0206, an engineered antimicrobial peptide, to address these limitations. PLG0206 has broad-spectrum activity against >1,200 multidrug-resistant (MDR) ESKAPEE clinical isolates, is rapidly bactericidal, and displays potent anti-biofilm activity against diverse MDR pathogens. PLG0206 displays activity in diverse animal infection models following both systemic (urinary tract infection) and local (prosthetic joint infection) administration. These findings support continuing clinical development of PLG0206 and validate use of rational design for peptide therapeutics to overcome limitations associated with difficult-to-drug pharmaceutical targets.


Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Biofilmes , Preparações Farmacêuticas
8.
Curr Rev Clin Exp Pharmacol ; 16(3): 263-272, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32778037

RESUMO

BACKGROUND: To address multidrug resistance, we developed engineered Cationic Antimicrobial Peptides (eCAPs). Lead eCAP WLBU2 displays potent activity against drug-resistant bacteria and effectively treats lethal bacterial infections in mice, reducing bacterial loads to undetectable levels in diverse organs. OBJECTIVE: To support the development of WLBU2, we conducted a mass balance study. METHODS: CD1 mice were administered 10, 15, 20 and 30 mg/kg of QDx5 WLBU2 or a single dose of [14C]-WLBU2 at 15 mg/kg IV. Tolerability, tissue distribution and excretion were evaluated with liquid scintillation and HPLC-radiochromatography. RESULTS: The maximum tolerated dose of WLBU2 is 20 mg/kg IV. We could account for greater than >96% of the radioactivity distributed within mouse tissues at 5 and 15 min. By 24h, only ~40-50% of radioactivity remained in the mice. The greatest % of the dose was present in liver, accounting for ~35% of radioactivity at 5 and 15 min, and ~ 8% of radioactivity remained at 24h. High radioactivity was also present in kidneys, plasma, red blood cells and lungs, while less than 0.2% of radioactivity was present in brain, fat, or skeletal muscle. Urinary and fecal excretion accounted for 12.5 and 2.2% of radioactivity at 24h. CONCLUSION: WLBU2 distributes widely to mouse tissues and is rapidly cleared with a terminal radioactivity half-life of 22 h, a clearance of 27.4 mL/h/kg, and a distribution volume of 0.94 L/kg. At 2-100 µg-eq/g, the concentrations of 14C-WLBU2 appear high enough in the tissues to account for the inhibition of microbial growth.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Infecções Bacterianas , Animais , Peptídeos Antimicrobianos , Radioisótopos de Carbono , Camundongos
9.
Immunol Res ; 36(1-3): 51-60, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17337766

RESUMO

The ultimate goal of an AIDS vaccine is to elicit potent cellular and humoral immune responses that will result in broadly enduring protective immunity. During the past several years, we have focused on characterizing the quantitative and qualitative properties of the antibody response, principally working to define the mechanism(s) of antibody-mediated neutralization in vitro. We have utilized a panel of monoclonal antibodies generated from monkeys infected with attenuated SIV for more than 8 mo to dissect the early events of virus infection involved in antibody-mediated neutralization. Presented herein are highlights from our studies that have identified potential mechanisms by which antibodies neutralize SIV in vitro.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Antivirais/sangue , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos/imunologia , HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Macaca mulatta , Testes de Neutralização , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia
10.
J Med Microbiol ; 65(6): 554-565, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27046192

RESUMO

We previously reported a series of de novo engineered cationic antibiotic peptides (eCAPs) consisting exclusively of arginine and tryptophan (WR) that display potent activity against diverse multidrug-resistant (MDR) bacterial strains. In this study, we sought to examine the influence of arginine compared to lysine on antibacterial properties by direct comparison of the WR peptides (8-18 residues) with a parallel series of engineered peptides containing only lysine and tryptophan. WR and WK series were compared for antibacterial activity by bacterial killing and growth inhibition assays and for mechanism of peptide-bacteria interactions by surface plasmon resonance and flow cytometry. Mammalian cytotoxicity was also assessed by flow cytometry, haemolytic and tetrazolium-based assays. The shortest arginine-containing peptides (8 and 10 mers) displayed a statistically significant increase in activity compared to the analogous lysine-containing peptides. The WR and WK peptides achieved maximum antibacterial activity at the 12-mer peptide (WK12 or WR12). Further examination of antibacterial mechanisms of the optimally active 12-mer peptides using surface plasmon resonance and flow cytometry demonstrates stronger interactions with Pseudomonasaeruginosa, greater membrane permeabilizing activity, and lower inhibitory effects of divalent cations on activity and membrane permeabilization properties of WR12 compared to WK12 (P < 0.05). Importantly, WK12 and WR12 displayed similar negligible haemolytic and cytotoxic effects at peptide concentrations up to ten times the MIC or 20 times the minimum bactericidal concentration. Thus, arginine, compared to lysine, can indeed yield enhanced antibacterial activity to minimize the required length to achieve functional antimicrobial peptides.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Arginina/química , Lisina/química , Pseudomonas aeruginosa/efeitos dos fármacos , Triptofano/química , Farmacorresistência Bacteriana Múltipla , Humanos , Macrófagos/efeitos dos fármacos , Ligação Proteica
11.
Expert Opin Biol Ther ; 14(1): 11-4, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24206062

RESUMO

Antibiotics have been among the most successful classes of therapeutics and have enabled many of modern medicine's greatest advances. However, antibiotic-resistant bacteria are emerging as critical public health threats, with recent accounts of bacterial strains resistant to all approved antibiotics. Antimicrobial peptides (AMPs) are naturally occurring molecules with the potential to serve as the basis for a new class of anti-infectives targeting these difficult-to-treat bacteria. The unique activities and features of AMPs are discussed, with a focus toward the clinical importance of priming the antibiotic pipeline and the role AMPs can fulfill in the future of fighting drug-resistant bacteria.


Assuntos
Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Desenho de Fármacos , Farmacorresistência Bacteriana , Humanos
12.
Viruses ; 6(1): 284-300, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24441863

RESUMO

Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env) displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.


Assuntos
Retroviridae/fisiologia , Proteínas do Envelope Viral/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Humanos , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/genética , Montagem de Vírus
13.
PLoS One ; 8(5): e65220, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23724133

RESUMO

Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20-50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.


Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Epitopos/química , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Testes de Neutralização , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Vírion/imunologia , Vírion/metabolismo
14.
PLoS One ; 5(12): e15261, 2010 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-21151874

RESUMO

The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.


Assuntos
Proteína gp41 do Envelope de HIV/química , HIV-1/metabolismo , Sequência de Aminoácidos , Separação Celular , Detergentes/farmacologia , Epitopos/química , Citometria de Fluxo , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Bicamadas Lipídicas/química , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Proteínas do Envelope Viral/química
15.
Virology ; 400(1): 86-92, 2010 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-20153009

RESUMO

Achieving humoral immunity against human immunodeficiency virus (HIV) is a major obstacle in AIDS vaccine development. Despite eliciting robust humoral responses to HIV, exposed hosts rarely produce broadly neutralizing antibodies. The present study utilizes simian immunodeficiency virus (SIV) to identify viral epitopes that conferred antibody neutralization to clone SIV/17E-CL, an in vivo variant derived from neutralization resistant SIVmac239. Neutralization assays using rhesus macaque monoclonal antibodies were performed on viruses engineered to express single or multiple amino acid mutations. Results identified a single amino acid mutation, P334R, in the carboxy-terminal half of the V3 loop as a critical residue that induced neutralization while retaining normal glycoprotein expression on the surface of the virus. Furthermore, the R334 residue yielded neutralization sensitivity by antibodies recognizing diverse conformational and linear epitopes of gp120, suggesting that neutralization phenotype was a consequence of global structural changes of the envelope protein rather than a specific site epitope.


Assuntos
Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Linhagem Celular , Epitopos/genética , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Humanos , Macaca mulatta , Mutagênese Sítio-Dirigida , Mutação , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
16.
PLoS One ; 3(1): e1501, 2008 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-18231588

RESUMO

BACKGROUND: Vaccination is a cost-effective counter-measure to the threat of seasonal or pandemic outbreaks of influenza. To address the need for improved influenza vaccines and alternatives to egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine. METHODOLOGY/PRINCIPAL FINDINGS: We generated from a baculovirus expression system using insect cells, a non-infectious recombinant VLP vaccine from both influenza A H5N1 clade 1 and clade 2 isolates with pandemic potential. VLPs were administered to mice in either a one-dose or two-dose regimen and the immune responses were compared to those induced by recombinant hemagglutinin (rHA). Both humoral and cellular responses were analyzed. Mice vaccinated with VLPs were protected against challenge with lethal reassortant viruses expressing the H5N1 HA and NA, regardless if the H5N1 clade was homologous or heterologous to the vaccine. However, rHA-vaccinated mice showed considerable weight loss and death following challenge with the heterovariant clade virus. Protection against death induced by VLPs was independent of the pre-challenge HAI titer or cell-mediated responses to HA or M1 since vaccinated mice, with low to undetectable cross-clade HAI antibodies or cellular responses to influenza antigens, were still protected from a lethal viral challenge. However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines. CONCLUSION/SIGNIFICANCE: This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate. The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.


Assuntos
Reações Cruzadas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Neuraminidase/imunologia , Vírion/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Baculoviridae , Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Imunidade Celular , Virus da Influenza A Subtipo H5N1/enzimologia , Vacinas contra Influenza/imunologia , Camundongos , Spodoptera , Ressonância de Plasmônio de Superfície , Vírion/isolamento & purificação
17.
J Virol ; 81(2): 465-73, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17079309

RESUMO

A feline immunodeficiency virus (FIV) provirus with a vif gene deletion (FIVDelta vifATGgamma) that coexpresses feline gamma interferon (IFN-gamma) was tested as a proviral DNA vaccine to extend previous studies showing efficacy with an FIV-pPPRDelta vif DNA vaccine. Cats were vaccinated with either FIVDelta vifATGgamma or FIV-pPPRDelta vif proviral plasmid DNA or with both FIV-pPPRDelta vif DNA and a feline IFN-gamma expression plasmid (pCDNA-IFNgamma). A higher frequency of FIV-specific T-cell proliferation responses was observed in cats immunized with either FIVDelta vifATGgamma or FIV-pPPRDelta vif plus pCDNA-IFNgamma, while virus-specific cytotoxic-T-lymphocyte responses were comparable between vaccine groups. Antiviral antibodies were not observed postvaccination. Virus-specific cellular and humoral responses were similar between vaccine groups after challenge with a biological FIV isolate (FIV-PPR) at 13 weeks postimmunization. All vaccinated and unvaccinated cats were infected after FIV-PPR challenge and exhibited similar plasma virus loads. Accordingly, inclusion of plasmids containing IFN-gamma did not enhance the efficacy of FIV-pPPRDelta vif DNA immunization. Interestingly, the lack of protection associated with FIV-pPPRDelta vif DNA immunization contrasted with findings from a previous study and suggested that multiple factors, including timing of FIV-pPPRDelta vif inoculations and challenge, as well as route of challenge virus delivery, may significantly impact vaccine efficacy.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Síndrome de Imunodeficiência Adquirida Felina/prevenção & controle , Vírus da Imunodeficiência Felina/imunologia , Interferon gama/administração & dosagem , Provírus , Vacinas Atenuadas/administração & dosagem , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Gatos , Vírus da Imunodeficiência Felina/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Provírus/genética , Provírus/imunologia , Linfócitos T/imunologia , Vacinação/veterinária , Vacinas Atenuadas/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
18.
J Virol ; 79(5): 2666-77, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15708986

RESUMO

Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAV(UK)DeltaS2 derived from a virulent proviral clone, EIAV(UK)DeltaS2/DU containing a second gene mutation in the virulent proviral clone, and EIAV(PR)DeltaS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 10(2) to 10(3) RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAV(PV) strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity.


Assuntos
Vírus da Anemia Infecciosa Equina/imunologia , Vacinas Virais/farmacologia , Animais , Anticorpos Antivirais/sangue , Anemia Infecciosa Equina/imunologia , Anemia Infecciosa Equina/prevenção & controle , Anemia Infecciosa Equina/virologia , Feminino , Genes Virais , Cavalos , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/patogenicidade , Masculino , Mutação , Fatores de Tempo , Vacinas Atenuadas/genética , Vacinas Atenuadas/farmacologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/genética
19.
J Virol ; 79(19): 12311-20, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16160158

RESUMO

Increasing evidence suggests that an effective AIDS vaccine will need to elicit both broadly reactive humoral and cellular immune responses. Potent and cross-reactive neutralization of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) by polyclonal and monoclonal antibodies is well documented. However, the mechanisms of antibody-mediated neutralization have not been defined. The current study was designed to determine whether the specificity and quantitative properties of antibody binding to SIV envelope proteins correlate with neutralization. Using a panel of rhesus monoclonal antibodies previously characterized for their ability to bind and neutralize variant SIVs, we compared the kinetic rates and affinity of antibody binding to soluble envelope trimers by using surface plasmon resonance. We identified significant differences in the kinetic rates but not the affinity of monoclonal antibody binding to the neutralization-sensitive SIV/17E-CL and neutralization-resistant SIVmac239 envelope proteins that correlated with the neutralization sensitivities of the corresponding virus strains. These results suggest for the first time that neutralization resistance may be related to quantitative differences in the rates but not the affinity of the antibody-envelope interaction and may provide one mechanism for the inherent resistance of SIVmac239 to neutralization in vitro. Further, we provide evidence that factors in addition to antibody binding, such as epitope specificity, contribute to the mechanisms of neutralization of SIV/17E-CL in vitro. This study will impact the method by which HIV/SIV vaccines are evaluated and will influence the design of candidate AIDS vaccines capable of eliciting effective neutralizing antibody responses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Antígenos Virais/imunologia , Cinética , Macaca mulatta , Testes de Neutralização , Ligação Proteica , Proteínas do Envelope Viral/imunologia
20.
J Virol ; 78(3): 1525-39, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14722307

RESUMO

One mechanism of immune evasion utilized by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins is the presence of a dense carbohydrate shield. Accumulating evidence from in vitro and in vivo experiments suggests that alterations in N-linked glycosylation of SIV gp120 can enhance host humoral immune responses that may be involved in immune control. The present study was designed to determine the ability of glycosylation mutant viruses to redirect antibody responses to shielded envelope epitopes. The influence of glycosylation on the maturation and specificity of antibody responses elicited by glycosylation mutant viruses containing mutations of specific N-linked sites in and near the V1 and V2 regions of SIVmac239 gp120 was determined. Results from these studies demonstrated a remarkably similar maturation of antibody responses to native, fully glycosylated envelope proteins. However, analyses of antibodies to defined envelope domains revealed that mutation of glycosylation sites in V1 resulted in increased antibody recognition to epitopes in V1. In addition, we demonstrated for the first time that mutation of glycosylation sites in V1 resulted in a redirection of antibody responses to the V3 loop. Taken together, these results demonstrate that N-linked glycosylation is a determinant of SIV envelope B-cell immunogenicity in addition to in vitro antigenicity. In addition, our results demonstrate that the absence of N-linked carbohydrates at specific sites can influence the exposure of epitopes quite distant in the linear sequence.


Assuntos
Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Glicoproteínas de Membrana/química , Mutação , Fragmentos de Peptídeos/imunologia , Vírus da Imunodeficiência Símia/imunologia , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Glicosilação , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Macaca mulatta , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
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