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Comparative methods in molecular evolution and structural biology rely heavily upon the site-wise analysis of DNA sequence and protein structure, both static forms of information. However, it is widely accepted that protein function results from nanoscale nonrandom machine-like motions induced by evolutionarily conserved molecular interactions. Comparisons of molecular dynamics (MD) simulations conducted between homologous sites representative of different functional or mutational states can potentially identify local effects on binding interaction and protein evolution. In addition, comparisons of different (i.e., nonhomologous) sites within MD simulations could be employed to identify functional shifts in local time-coordinated dynamics indicative of logic gating within proteins. However, comparative MD analysis is challenged by the large fraction of protein motion caused by random thermal noise in the surrounding solvent. Therefore, properly denoised MD comparisons could reveal functional sites involving these machine-like dynamics with good accuracy. Here, we introduce ATOMDANCE, a user-interfaced suite of comparative machine learning-based denoising tools designed for identifying functional sites and the patterns of coordinated motion they can create within MD simulations. ATOMDANCE-maxDemon4.0 employs Gaussian kernel functions to compute site-wise maximum mean discrepancy between learned features of motion, thereby assessing denoised differences in the nonrandom motions between functional or evolutionary states (e.g., ligand bound versus unbound, wild-type versus mutant). ATOMDANCE-maxDemon4.0 also employs maximum mean discrepancy to analyze potential random amino acid replacements allowing for a site-wise test of neutral versus nonneutral evolution on the divergence of dynamic function in protein homologs. Finally, ATOMDANCE-Choreograph2.0 employs mixed-model analysis of variance and graph network to detect regions where time-synchronized shifts in dynamics occur. Here, we demonstrate ATOMDANCE's utility for identifying key sites involved in dynamic responses during functional binding interactions involving DNA, small-molecule drugs, and virus-host recognition, as well as understanding shifts in global and local site coordination occurring during allosteric activation of a pathogenic protease.
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Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (â¼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2-/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity.IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.
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Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteção Cruzada , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Fatores Imunológicos/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos , Modelos Animais de Doenças , Fatores Imunológicos/administração & dosagem , Camundongos , Resultado do TratamentoRESUMO
Subcellular biomolecular localization is critical for the metabolic and structural properties of the cell. The functional implications of the spatiotemporal distribution of protein complexes during the bacterial cell cycle have long been acknowledged; however, the molecular mechanisms for generating and maintaining their dynamic localization in bacteria are not completely understood. Here we demonstrate that the trans-envelope Tol-Pal complex, a widely conserved component of the cell envelope of Gram-negative bacteria, is required to maintain the polar positioning of chemoreceptor clusters in Escherichia coli. Localization of the chemoreceptors was independent of phospholipid composition of the membrane and the curvature of the cell wall. Instead, our data indicate that chemoreceptors interact with components of the Tol-Pal complex and that this interaction is required to polarly localize chemoreceptor clusters. We found that disruption of the Tol-Pal complex perturbs the polar localization of chemoreceptors, alters cell motility, and affects chemotaxis. We propose that the E. coli Tol-Pal complex restricts mobility of the chemoreceptor clusters at the cell poles and may be involved in regulatory mechanisms that co-ordinate cell division and segregation of the chemosensory machinery.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
The HIV-1 protease is one of several common key targets of combination drug therapies for human immunodeficiency virus infection and acquired immunodeficiency syndrome. During the progression of the disease, some individual patients acquire drug resistance due to mutational hotspots on the viral proteins targeted by combination drug therapies. It has recently been discovered that drug-resistant mutations accumulate on the "flap region" of the HIV-1 protease, which is a critical dynamic region involved in nonspecific polypeptide binding during invasion and infection of the host cell. In this study, we utilize machine learning-assisted comparative molecular dynamics, conducted at single amino acid site resolution, to investigate the dynamic changes that occur during functional dimerization and drug binding of wild-type and common drug-resistant versions of the main protease. We also use a multiagent machine learning model to identify conserved dynamics of the HIV-1 main protease that are preserved across simian and feline protease orthologs. We find that a key conserved functional site in the flap region, a solvent-exposed isoleucine (Ile50) that controls flap dynamics is functionally targeted by drug resistance mutations, leading to amplified molecular dynamics affecting the functional ability of the flap region to hold the drugs. We conclude that better long-term patient outcomes may be achieved by designing drugs that target protease regions that are less dependent upon single sites with large functional binding effects.
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Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens' eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.
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Widespread human transmission of SARS-CoV-2 highlights the substantial public health, economic and societal consequences of virus spillover from wildlife and also presents a repeated risk of reverse spillovers back to naive wildlife populations. We employ comparative statistical analyses of a large set of short-term molecular dynamic (MD) simulations to investigate the potential human-to-bat (genus Rhinolophus) cross-species infectivity allowed by the binding of SARS-CoV-2 receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) across the bat progenitor strain and emerging human strain variants of concern (VOC). We statistically compare the dampening of atom motion across protein sites upon the formation of the RBD/ACE2 binding interface using various bat versus human target receptors (i.e. bACE2 and hACE2). We report that while the bat progenitor viral strain RaTG13 shows some pre-adaption binding to hACE2, it also exhibits stronger affinity to bACE2. While early emergent human strains and later VOCs exhibit robust binding to both hACE2 and bACE2, the delta and omicron variants exhibit evolutionary adaption of binding to hACE2. However, we conclude there is a still significant risk of mammalian cross-species infectivity of human VOCs during upcoming waves of infection as COVID-19 transitions from a pandemic to endemic status.
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The identification of viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a molecular dynamics (MD)-based approach that goes beyond contact mapping, scales well to a desktop computer with a modern graphics processor, and enables the user to identify functional protein sites that are prone to vaccine escape in a viral antigen. We first implement our MD pipeline to employ site-wise calculation of Kullback-Leibler divergence in atom fluctuation over replicate sets of short-term MD production runs thus enabling a statistical comparison of the rapid motion of influenza hemagglutinin (HA) in both the presence and absence of three well-known neutralizing antibodies. Using this simple comparative method applied to motions of viral proteins, we successfully identified in silico all previously empirically confirmed sites of escape in influenza HA, predetermined via selection experiments and neutralization assays. Upon the validation of our computational approach, we then surveyed potential hotspot residues in the receptor binding domain of the SARS-CoV-2 virus in the presence of COVOX-222 and S2H97 antibodies. We identified many single sites in the antigen-antibody interface that are similarly prone to potential antibody escape and that match many of the known sites of mutations arising in the SARS-CoV-2 variants of concern. In the Omicron variant, we find only minimal adaptive evolutionary shifts in the functional binding profiles of both antibodies. In summary, we provide an inexpensive and accurate computational method to monitor hotspots of functional evolution in antibody binding footprints.
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Comparative analysis of protein structure or sequence alignments often ignores the protein dynamics and function. We offer a graphical user interface to a computing pipeline, complete with molecular visualization, enabling the biophysical simulation and statistical comparison of two-state functional protein dynamics (i.e., single unbound state vs. complex with a ligand, DNA, or protein). We utilize multi-agent machine learning classifiers to identify functionally conserved dynamic motions and compare them in genetic or drug-class variants. For complete details on the use and execution of this profile, please refer to Babbitt et al. (2020b, 2020a, 2018) and Rynkiewicz et al. (2021).
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Proteínas , Software , Simulação por Computador , Aprendizado de Máquina , Proteínas/química , Alinhamento de SequênciaRESUMO
The influenza virus neuraminidase (NA) is primarily involved in the release of progeny viruses from infected cells-a critical role for virus replication. Compared to the immuno-dominant hemagglutinin, there are fewer NA subtypes, and NA experiences a slower rate of antigenic drift and reduced immune selection pressure. Furthermore, NA inhibiting antibodies prevent viral egress, thus preventing viral spread. Anti-NA immunity can lessen disease severity, reduce viral shedding, and decrease viral lung titers in humans and various animal models. As a result, there has been a concerted effort to investigate the possibilities of incorporating immunogenic forms of NA as a vaccine antigen in future vaccine formulations. In this review, we discuss NA-based immunity and describe several human NA-specific monoclonal antibodies (mAbs) that have a broad range of protection. We also review vaccine platforms that are investigating NA antigens in pre-clinical models and their potential use for next-generation influenza virus vaccines. The evidence presented here supports the inclusion of immunogenic NA in future influenza virus vaccines.
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The influenza virus neuraminidase (NA) plays an integral role in the influenza virus life cycle through the release of virions from infected cells. NA-specific antibodies can impede virus replication by binding to the NA and blocking its enzymatic activity, providing significant protection from influenza-associated morbidity and mortality. NA included in current seasonal influenza virus vaccines exhibits low immunogenicity, potentially caused by compromised antigenic integrity during vaccine production. To determine how certain types of "stress" could influence the antigenicity of NA we performed a series of in vitro experiments where we treated NA with formalin, EDTA or heat and measured the impact of these treatments on NA enzymatic activity and structural integrity. We found that increasing concentrations of formalin or EDTA and increasing temperature abolished the enzymatic activity of both H1N1, H3N2, and influenza B purified viruses and recombinant NA proteins. However, formalin and EDTA treatment did not drastically affect conformational epitopes found on the NA, whereas heat treatment abolished conformational epitopes. We next performed a vaccination experiment, where mice were vaccinated with recombinant N2 NA treated with 0.3% formalin or 0.125 M EDTA (which both inactivated NA activity) were protected from virus challenge while animals vaccinated with heat treated NA were not. We next tested the protective effect of monomeric (no enzymatic activity) versus tetrameric (highly active) N1 NA. Again, only the tetrameric form protected mice from challenge while the monomeric form did not. Together, our data demonstrate that enzymatically active NA is not required to induce protective antibody responses as a vaccine, however a correctly folded NA is essential.
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Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Animais , Anticorpos Antivirais , Formação de Anticorpos , Vírus da Influenza A Subtipo H3N2 , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase , Infecções por Orthomyxoviridae/prevenção & controleRESUMO
The standard method to quantify the hemagglutinin content of influenza virus vaccines is the single radial immunodiffusion assay. This assay primarily relies on polyclonal antibodies against the head domain of the influenza virus hemagglutinin, which is the main target antigen of influenza virus vaccines. Novel influenza virus vaccine candidates that redirect the immune response towards the evolutionary more conserved hemagglutinin stalk, including chimeric hemagglutinin and headless hemagglutinin constructs, are highly dependent on the structural integrity of the protein to present conformational epitopes for neutralizing antibodies. In this study, we describe a novel enzyme-linked immunosorbent assay that allows quantifying the amount of hemagglutinin with correctly folded stalk domains and which could be further developed into a potency assay for stalk-based influenza virus vaccines.
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Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Vacinas contra Influenza/análise , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/químicaRESUMO
Bacterial cell division requires identification of the division site, assembly of the division machinery, and constriction of the cell envelope. These processes are regulated in response to several cellular and environmental signals. Here, we use small molecule iron chelators to characterize the surprising connections between bacterial iron homeostasis and cell division. We demonstrate that iron starvation downregulates the transcription of genes encoding proteins involved in cell division, reduces protein biosynthesis, and prevents correct positioning of the division machinery at the division site. These combined events arrest the constriction of the cell during late stages of cytokinesis in a manner distinct from known mechanisms of inhibiting cell division. Overexpression of genes encoding cell division proteins or iron transporters partially suppresses the biological activity of iron chelators and restores growth and division. We propose a model demonstrating the effect of iron availability on the regulatory mechanisms coordinating division in response to the nutritional state of the cell.
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Bactérias/citologia , Benzimidazóis/farmacologia , Hidrazinas/farmacologia , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Naftalenos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzimidazóis/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/metabolismo , Cobalto/farmacologia , Cobre/farmacologia , Citocinese/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Hidrazinas/metabolismo , Ferro/farmacologia , Quelantes de Ferro/metabolismo , Naftalenos/metabolismo , Peptidoglicano/metabolismoRESUMO
Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for "original antigenic sin." Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.)IMPORTANCE Anti-neuraminidase antibodies can afford broad protection from influenza virus infection in animal models and humans. However, little is known about the breadth and magnitude of the anti-neuraminidase response in the human population. Here we assessed antibody levels of children, adults, and the elderly against a panel of N1, N2, and type B influenza virus neuraminidases. We demonstrated that antibody levels measured by ELISA correlate well with functional neuraminidase inhibition titers. This is an important finding since ELISA is a simpler method than functional assays and can be implemented in high-throughput settings to analyze large numbers of samples. Furthermore, we showed that low titers of broadly cross-reactive antibodies against neuraminidase are prevalent in humans. By the use of an appropriate vaccination strategy, these titers could potentially be boosted to levels that might provide broad protection from influenza virus infection.
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Anticorpos Antivirais/sangue , Neuraminidase/imunologia , Proteínas Virais/imunologia , Adolescente , Adulto , Filhos Adultos , Fatores Etários , Idoso , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Lactente , Pessoa de Meia-Idade , Neuraminidase/análise , Ensaios Clínicos Controlados Aleatórios como Assunto , Adulto JovemRESUMO
The immunogenicity of current influenza virus vaccines is assessed by measuring an increase of influenza virus-specific antibodies in a hemagglutination inhibition assay. This method exclusively measures antibodies against the hemagglutinin head domain. While this domain is immunodominant, it has been shown that hemagglutination inhibition titers do not always accurately predict protection from disease. In addition, several novel influenza virus vaccines that are currently under development do not target the hemagglutinin head domain, but rather more conserved sites, including the hemagglutinin stalk. Importantly, antibodies against the hemagglutinin stalk do not show activity in hemagglutination inhibition assays and will require different methods for quantification. In this study, we tested human serum samples from a seasonal influenza virus vaccination trial and an avian H5N1 virus vaccination trial for antibody activities in multiple types of assays, including binding assays and also functional assays. We then performed serum transfer experiments in mice which then received an H1N1 virus challenge to assess the in vivo protective effects of the antibodies. We found that hemagglutinin-specific antibody levels measured in an enzyme-linked immunosorbent assay (ELISA) correlated well with protection from weight loss in mice. In addition, we found that weight loss was also inversely correlated with the level of serum antibody-dependent cellular cytotoxicity (ADCC) as measured in a reporter assay. These findings indicate that protection is in part conferred by Fc-dependent mechanisms. In conclusion, ELISAs can be used to measure hemagglutinin-specific antibody levels that could serve as a surrogate marker of protection for universal influenza virus vaccines.IMPORTANCE Influenza viruses are a serious concern for public health and cause a large number of deaths worldwide every year. Current influenza virus vaccines can confer protection from disease, but they often show low efficacy due to the ever-changing nature of the viruses. Novel vaccination approaches target conserved epitopes of the virus, including the hemagglutinin stalk domain, to elicit universally protective antibodies that also bind to mutated viruses or new subtypes of viruses. Importantly, the hemagglutination inhibition assay-the only assay that has been accepted as a correlate of protection by regulatory authorities-cannot measure antibodies against the hemagglutinin stalk domain. Therefore, novel correlates of protection and assays to measure vaccine immunogenicity need to be developed. In this study, we correlated the results from multiple assays with protection in mice after transfer of human serum and a lethal virus challenge to investigate potential novel serological surrogate markers for protection.
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Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização Passiva , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/imunologia , Adulto , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Citotoxicidade Celular Dependente de Anticorpos , Biomarcadores , Ensaios Clínicos como Assunto , Reações Cruzadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Masculino , Camundongos , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , VacinaçãoRESUMO
Influenza viruses evade human adaptive immune responses due to continuing antigenic changes. This makes it necessary to re-formulate and re-administer current seasonal influenza vaccines on an annual basis. Our pan-influenza vaccination approach attempts to redirect antibody responses from the variable, immuno-dominant hemagglutinin head towards the conserved-but immuno-subdominant-hemagglutinin stalk. The strategy utilizes sequential immunization with chimeric hemagglutinin-based vaccines expressing exotic head domains, and a conserved hemagglutinin stalk. We compared a live-attenuated influenza virus prime followed by an inactivated split-virus boost to two doses of split-virus vaccines and assessed the impact of adjuvant on protection against challenge with pandemic H1N1 virus in ferrets. All tested immunization regimens successfully induced broadly cross-reactive antibody responses. The combined live-attenuated/split virus vaccination conferred superior protection against pandemic H1N1 infection compared to two doses of split-virus vaccination. Our data support advancement of this chimeric hemagglutinin-based vaccine approach to clinical trials in humans.
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Bacterial DNA gyrase is an essential type II topoisomerase that enables cells to overcome topological barriers encountered during replication, transcription, recombination, and repair. This enzyme is ubiquitous in bacteria and represents an important clinical target for antibacterial therapy. In this paper we report the characterization of three exciting new gyramide analogs-from a library of 183 derivatives-that are potent inhibitors of DNA gyrase and are active against clinical strains of gram-negative bacteria (Escherichia coli, Shigella flexneri, and Salmonella enterica; 3 of 10 wild-type strains tested) and gram-positive bacteria (Bacillus spp., Enterococcus spp., Staphylococcus spp., and Streptococcus spp.; all 9 of the wild-type strains tested). E. coli strains resistant to the DNA gyrase inhibitors ciprofloxacin and novobiocin display very little cross-resistance to these new gyramides. In vitro studies demonstrate that the new analogs are potent inhibitors of the DNA supercoiling activity of DNA gyrase (IC50s of 47-170 nM) but do not alter the enzyme's ATPase activity. Although mutations that confer bacterial cells resistant to these new gyramides map to the genes encoding the subunits of the DNA gyrase (gyrA and gyrB genes), overexpression of GyrA, GyrB, or GyrA and GyrB together does not suppress the inhibitory effect of the gyramides. These observations support the hypothesis that the gyramides inhibit DNA gyrase using a mechanism that is unique from other known inhibitors.