Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells... more Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we describe an assembly-line approach to build AVVs by engineering the well-characterized structural components of bacteriophage T4. Starting with a 120 × 86 nm capsid shell that can accommodate 171-Kbp DNA and thousands of protein copies, various combinations of biomolecules, including DNAs, proteins, RNAs, and ribonucleoproteins, are externally and internally incorporated. The nanoparticles are then coated with cationic lipid to enable efficient entry into human cells. As proof of concept, we assemble a series of AVVs designed to deliver full-length dystrophin gene or perform various molecular operations to remodel human genome, including genome editing, gene recombination, gene replacement, gene expression, and gene silencing. These large capacity, customizable, multiplex, and all-in-one phage-based AVVs represent an...
IntroductionInfants acquire maternal antibodies by Fc receptor transcytosis across the placenta d... more IntroductionInfants acquire maternal antibodies by Fc receptor transcytosis across the placenta during pregnancy. Fc receptors are expressed on immune cells and are important for activation of effector cell functions.MethodsIn this study, we evaluated Fc receptor engagement and ADCC activity of plasma binding antibodies from human immunodeficiency virus-1 (HIV) -infected mothers and to identify factors that may contribute to protection from HIV vertical transmission.ResultsHIV-specific binding and Fc receptor engagement of plasma antibodies varied between mothers by transmission status and infants by infection status. Non-transmitting (NT) mothers and HIV-uninfected infants had antibodies with higher neonatal Fc receptor (FcRn) and FcγR engagement, as compared to transmitting (T) mothers and HIV+ infants, respectively. A significant inverse correlation between plasma antibody FcRn and FcγR engagement was observed for T mothers, but not NT mothers. Conversely, a significant direct co...
Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry... more Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry sites are considered to be the most effective way to prevent infection. A universal platform that can be customized for development of mucosal vaccines against any given pathogen is therefore highly desired. Here, we demonstrate an efficient approach to develop nasal mucosal vaccines through genetic engineering of T4 phage to generate antigen-decorated nanoparticles. The antigen coding sequence was inserted into T4 genome in-frame at the C terminus of Soc (small outer capsid protein) using the CRISPR-Cas phage editing technology. During the propagation of recombinant T4 phages in E. coli, the Soc-antigen fusion proteins self-assemble on T4 capsids to form antigen-decorated nanoparticles that have intrinsic adjuvant activity and mucosal adhesive property. As a proof of concept, we showed that intranasal immunization with Flu viral M2e-decorated T4 nanoparticles efficiently induced local ...
The Corona Virus Disease 2019 (COVID-19) pandemic has huge impacts on the world, including human ... more The Corona Virus Disease 2019 (COVID-19) pandemic has huge impacts on the world, including human health and economic decline. The COVID-19 has severe infectivity, especially the elderly with chronic diseases will cause various complications after infection and accelerate the disease process. In addition, COVID-19 will also affect their mental health. Therefore, the mental health of elderly patients with chronic diseases cannot be ignored. The aim of this study was to investigate the well-being level of elderly people with chronic disease during COVID-19 postpandemic period in Beijing and analysis related influencing factors, so as to provide a basis for improving the well-being level of elderly chronic patients during the postpandemic period. Elderly patients with chronic diseases who met the inclusion criteria in 5 different administrative regions in Beijing were selected to carry out a questionnaire survey. The contents of the questionnaire included general data, the Memorial Univ...
The latent HIV-1 reservoir containing stably integrated and transcriptionally silent proviruses i... more The latent HIV-1 reservoir containing stably integrated and transcriptionally silent proviruses in CD4+ T cells is a major barrier for virus eradication. Targeted reactivation of the latent reservoir remains a major challenge in establishing a path for an HIV-1 cure. Here, we investigated the possibility of reactivating the HIV-1 reservoir by targeting engineered bacteriophage T4 capsid nanoparticles to reservoir cells. The surface lattice of the 120 x 86 nm phage capsid was arrayed with CD4 binding ligands such as recombinant CD4DARPin or the HIV-1 gp140 envelope protein. When exposed to either PBMCs or the resting CD4+ T cellsin vitro, these nanoparticles caused T cells activation without inducing global T cell activation. Furthermore, the nanoparticles reactivated HIV-1 proviral transcription that led to virus assembly and release in the J-Lat cells, a cell line model of HIV-1 latency. Intriguingly, the observed T cell activation and HIV-1 latency reversal did not occur through t...
A CRISPR-engineered bacteriophage T4-COVID vaccine delivers multiple SARS-CoV-2 antigens for pote... more A CRISPR-engineered bacteriophage T4-COVID vaccine delivers multiple SARS-CoV-2 antigens for potential broader protection.
A new bacteriophage T4-induced DNA-dependent ATPase-endonuclease was purified to essential homoge... more A new bacteriophage T4-induced DNA-dependent ATPase-endonuclease was purified to essential homogeneity from an extract of late infected Escherichia coli. Both DNA-dependent ATPase and endonuclease activities co-chromatograph, co-sediment, and have been renatured from a single 43-kilodalton protein eluted following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that both activities are exerted by one multifunctional protein. Duplex, single-stranded, and supercoiled DNAs are all effective activators of the high specific activity ATPase which produces ADP and inorganic PO4. The enzyme displays a broad specificity towards the nucleoside and deoxynucleoside triphosphates, and the ATPase activity is strongly inhibited by DNA-intercalating compounds. The endonuclease appears to be most active on supercoiled DNA, producing double-stranded breaks in duplex DNA, and does not require nucleoside triphosphates. An antiserum against the purified enzyme immunoprecipitated it, inhibited its ATPase activity, and also precipitated from extracts a T4-induced protein of Mr = 43,000. This antigen was not found in uninfected E. coli, or following a gene 55am mutant (late protein synthesis defective) infection, and was not detected following infection with T4 amber mutants of any early capsid protein gene which blocks T4 head protein cleavage in vivo. In a pulse-chase experiment, the radioactive antigen was not found following a pulse of radioactive amino acids, but appeared after a chase with excess nonradioactive amino acids. The enzyme-related antigen is apparently produced by cleavage of a precursor by the T4 head assembly proteinase which processes a number of prohead proteins. These processing reactions are dependent in vivo upon assembly of the prohead and are required for its maturation. The evidence suggests that this enzyme functions in head assembly and DNA packaging, and originates as the cleavage product of a prohead precursor protein.
The objective of this study was to explore the efficacy of low dose of oxcarbazepine (OXC) in adu... more The objective of this study was to explore the efficacy of low dose of oxcarbazepine (OXC) in adult patients with newly diagnosed partial epilepsy in an actual clinical setting. The associated factors influencing the poor control of seizures were also evaluated. The epilepsy database (2010-2014) from the Epilepsy Clinic of West China Hospital was retrospectively reviewed. A total of 102 adult patients with newly diagnosed, previously untreated partial epilepsy initially treated with OXC were included, and divided into good response group (64) and poor response group (38) according to whether they were seizure-free for at least 12 months. There were 27 (26.5%) patients becoming seizure-free with OXC 600mg/day monotherapy. The remaining 75 patients had doses of either increasing OXC to 900mg/day (n=59) or the addition of another antiepileptic drug (AED) (n=16), with another 20 (19.6%) and six (5.9%) patients becoming seizure-free, respectively (P=0.788). In addition, two (2.0%) and ni...
Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuro... more Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuronal cells and efficiently evade the immune system. It has been a major medical challenge to eradicate them and, despite intensive efforts, an effective vaccine is not available. We previously showed that upon infection of antigen-presenting cells, HSV type 1 (HSV-1) rapidly and efficiently downregulates the major histocompatibility complex class I-like antigen-presenting molecule, CD1d, and potently inhibits its recognition by CD1d-restricted natural killer T (NKT) cells. It suppresses CD1d expression primarily by inhibiting its recycling to the cell surface after endocytosis. We identify here the viral glycoprotein B (gB) as the predominant CD1d-interacting protein. gB initiates the interaction with CD1d in the endoplasmic reticulum and stably associates with it throughout CD1d trafficking. However, an additional HSV-1 component, the serine-threonine kinase US3, is required for optimal ...
Studies on human immunodeficiency virus type 1 (HIV-1) diversity are critical for understanding v... more Studies on human immunodeficiency virus type 1 (HIV-1) diversity are critical for understanding viral pathogenesis and the emergence of immune escape variants and for design of vaccine strategies. To investigate HIV-1 population genetics, we used single-genome sequencing to obtain pro-pol and env sequences from longitudinal samples ( n = 93) from 14 acutely or recently infected patients. The first available sample after infection for 12/14 patients revealed HIV-1 populations with low genetic diversity, consistent with transmission or outgrowth of a single variant. In contrast, two patients showed high diversity and coexistence of distinct virus populations in samples collected days after a nonreactive enzyme-linked immunosorbent assay or indeterminate Western blot, consistent with transmission or outgrowth of multiple variants. Comparison of PR and RT sequences from the first sample for all patients with the consensus subgroup B sequence revealed that nearly all nonsynonymous differ...
The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A s... more The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A single structural protein comprises this dodecameric ring substructure of the prohead. Assembly of the phage T4 prohead occurs on the cytoplasmic membrane through a specific attachment at or near the gp20 DNA entrance vertex. An auxiliary head assembly gene product, gp40, was hypothesized to be involved in assembling the gp20 substructure. T4 genes 20,40 and 20 + 40 were cloned into expression vectors under lzpL promoter control. The corresponding T4 gene products were synthesized in high yield and were active as judged by their ability to complement the corresponding infecting T4 mutants in vivo. The cloned T4 gene 20 and gene 40 products were inserted into the cytoplasmic membrane as integral membrane proteins; however, gp20 was inserted into the membrane only when gp40 was also synthesized, whereas gp40 was inserted in the presence or absence of gp20. The gp20 insertion required a membrane potential, was not dependent upon the Escherichia coli groE gene, and assumed a defined membrane-spanning conformation, as judged by specific protease fragments protected by the membrane. The inserted gp20 structure could be probed by antibody binding and protein A-gold immunoelectron microscopy. The dat'a suggest that a specific gp20-gp4Gmembrane insertion structure constitutes the T4 prohead assembly initiation complex.
Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer... more Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer alternatives to whole pathogen vaccines. However, the antigens by themselves are not sufficiently immunogenic and require additives known as adjuvants to enhance immunogenicity and protective efficacy. Assembly of the antigens into virus-like nanoparticles (VLPs) is a better approach as it allows presentation of the epitopes in a more native context. The repetitive, symmetrical, and high density display of antigens on the VLPs mimic pathogen-associated molecular patterns seen on bacteria and viruses. The antigens, thus, might be better presented to stimulate host's innate as well as adaptive immune systems thereby eliciting both humoral and cellular immune responses. Bacteriophages such as phage T4 provide excellent platforms to generate the nanoparticle vaccines. The T4 capsid containing two non-essential outer proteins Soc and Hoc allow high density array of antigen epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Co-delivery of DNAs, targeting molecules, and/or molecular adjuvants provides additional advantages. Recent studies demonstrate that the phage T4 VLPs are highly immunogenic, do not need an adjuvant, and provide complete protection against bacterial and viral pathogens. Thus, phage T4 could potentially be developed as a "universal" VLP platform to design future multivalent vaccines against complex and emerging pathogens.
<p>(<b>A</b>) Schematic of native F1-V, F1mut-V and F1mut-V10. Cyan represents ... more <p>(<b>A</b>) Schematic of native F1-V, F1mut-V and F1mut-V10. Cyan represents the coding sequence of V antigen, and yellow, the putative immunomodulatory sequence that is part of V sequence. Rest of the colors represents the same as described in legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g002" target="_blank">Figure 2</a>. (<b>B</b>) Expression and solubility analysis of F1-V constructs were performed using the B-PER reagent. The samples were analyzed by SDS-PAGE and Coomassie blue staining. The positions of the F1-V protein bands are marked with red arrows. S, soluble fraction (supernatant from 12,000 <i>g</i> centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (<b>C</b>) F1-V, F1mut-V and F1mut-V10 were purified by HisTrap column chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. The calibration graph was generated by passing various molecular weight standards through the same column [Thyroglobulin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), aldolase (158 kDa), Ovalbumin (43 kDa), RNase A (14 kDa), and Albumin (67 kDa)]. The insert shows the purity of F1-V, F1mut-V, and F1mut-V10 proteins following SDS-PAGE and Coomassie blue staining of the peak fractions. The color of arrows corresponds to the color of the elution profiles of various proteins. (<b>D</b>) Stability of F1-V and F1mut-V proteins was tested by treatment with increasing amounts of trypsin at room temperature overnight. The ratios shown above the gel correspond to the ratios of F1-V or F1mut-V proteins to trypsin (wt∶wt). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details.</p
<p>The immunogenicity and protective efficacy of F1mut-V and other plague immunogens were e... more <p>The immunogenicity and protective efficacy of F1mut-V and other plague immunogens were evaluated in a mouse model. (<b>A</b>) Balb/c mice, twelve per group, were vaccinated with various plague antigens adjuvanted with alhydrogel. (<b>B</b>) Immunization scheme. (<b>C</b>) Antigen-specific antibody (IgG) titers were determined by ELISA, using purified V (I), F1mut2 (II), or YscF35/67 (III) as the coating antigen. No significant cross-reactivity was observed between the antibodies produced against one plague antigen versus a different plague antigen that was coated on the ELISA plate. Error bars represent S.D. “***” denotes p<0.001 (ANOVA). (<b>D</b>) Survival of immunized mice against intranasal challenge with 90 LD<sub>50</sub> of <i>Y. pestis</i> CO92. The survived mice were re-challenged with 9,800 LD<sub>50</sub> at day-48 post-first challenge. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.</p
<p>(<b>IgG1) and T<sub>H</sub>2 (IgG2a) responses.</b> The immuniza... more <p>(<b>IgG1) and T<sub>H</sub>2 (IgG2a) responses.</b> The immunization scheme is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g006" target="_blank">Figure 6B</a>. Seven days after the second boost, sera were collected and IgG1 (<b>A</b>) and IgG2a (<b>B</b>) titers were determined by ELISA. F1mut-V was used as the coating antigen, since it covers all the epitopes present in both F1mut-V and F1mut-V10. Note that the sera of the control T4 phage-immunized mice showed higher background. This was because T4 phage, as demonstrated in previous studies, induces a strong antibody response to its components. Consequently, the sera from T4 phage- immunized mice will have increased amounts of IgGs compared to the pre-immune sera, giving more non-specific background at low dilutions of the sera. Data shown are the antibody titers of 12 mice in each group with S.D. (error bars). *, p<0.05; ***, p<0.001 (ANOVA).</p
Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures ... more Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding th...
Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells... more Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we describe an assembly-line approach to build AVVs by engineering the well-characterized structural components of bacteriophage T4. Starting with a 120 × 86 nm capsid shell that can accommodate 171-Kbp DNA and thousands of protein copies, various combinations of biomolecules, including DNAs, proteins, RNAs, and ribonucleoproteins, are externally and internally incorporated. The nanoparticles are then coated with cationic lipid to enable efficient entry into human cells. As proof of concept, we assemble a series of AVVs designed to deliver full-length dystrophin gene or perform various molecular operations to remodel human genome, including genome editing, gene recombination, gene replacement, gene expression, and gene silencing. These large capacity, customizable, multiplex, and all-in-one phage-based AVVs represent an...
IntroductionInfants acquire maternal antibodies by Fc receptor transcytosis across the placenta d... more IntroductionInfants acquire maternal antibodies by Fc receptor transcytosis across the placenta during pregnancy. Fc receptors are expressed on immune cells and are important for activation of effector cell functions.MethodsIn this study, we evaluated Fc receptor engagement and ADCC activity of plasma binding antibodies from human immunodeficiency virus-1 (HIV) -infected mothers and to identify factors that may contribute to protection from HIV vertical transmission.ResultsHIV-specific binding and Fc receptor engagement of plasma antibodies varied between mothers by transmission status and infants by infection status. Non-transmitting (NT) mothers and HIV-uninfected infants had antibodies with higher neonatal Fc receptor (FcRn) and FcγR engagement, as compared to transmitting (T) mothers and HIV+ infants, respectively. A significant inverse correlation between plasma antibody FcRn and FcγR engagement was observed for T mothers, but not NT mothers. Conversely, a significant direct co...
Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry... more Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry sites are considered to be the most effective way to prevent infection. A universal platform that can be customized for development of mucosal vaccines against any given pathogen is therefore highly desired. Here, we demonstrate an efficient approach to develop nasal mucosal vaccines through genetic engineering of T4 phage to generate antigen-decorated nanoparticles. The antigen coding sequence was inserted into T4 genome in-frame at the C terminus of Soc (small outer capsid protein) using the CRISPR-Cas phage editing technology. During the propagation of recombinant T4 phages in E. coli, the Soc-antigen fusion proteins self-assemble on T4 capsids to form antigen-decorated nanoparticles that have intrinsic adjuvant activity and mucosal adhesive property. As a proof of concept, we showed that intranasal immunization with Flu viral M2e-decorated T4 nanoparticles efficiently induced local ...
The Corona Virus Disease 2019 (COVID-19) pandemic has huge impacts on the world, including human ... more The Corona Virus Disease 2019 (COVID-19) pandemic has huge impacts on the world, including human health and economic decline. The COVID-19 has severe infectivity, especially the elderly with chronic diseases will cause various complications after infection and accelerate the disease process. In addition, COVID-19 will also affect their mental health. Therefore, the mental health of elderly patients with chronic diseases cannot be ignored. The aim of this study was to investigate the well-being level of elderly people with chronic disease during COVID-19 postpandemic period in Beijing and analysis related influencing factors, so as to provide a basis for improving the well-being level of elderly chronic patients during the postpandemic period. Elderly patients with chronic diseases who met the inclusion criteria in 5 different administrative regions in Beijing were selected to carry out a questionnaire survey. The contents of the questionnaire included general data, the Memorial Univ...
The latent HIV-1 reservoir containing stably integrated and transcriptionally silent proviruses i... more The latent HIV-1 reservoir containing stably integrated and transcriptionally silent proviruses in CD4+ T cells is a major barrier for virus eradication. Targeted reactivation of the latent reservoir remains a major challenge in establishing a path for an HIV-1 cure. Here, we investigated the possibility of reactivating the HIV-1 reservoir by targeting engineered bacteriophage T4 capsid nanoparticles to reservoir cells. The surface lattice of the 120 x 86 nm phage capsid was arrayed with CD4 binding ligands such as recombinant CD4DARPin or the HIV-1 gp140 envelope protein. When exposed to either PBMCs or the resting CD4+ T cellsin vitro, these nanoparticles caused T cells activation without inducing global T cell activation. Furthermore, the nanoparticles reactivated HIV-1 proviral transcription that led to virus assembly and release in the J-Lat cells, a cell line model of HIV-1 latency. Intriguingly, the observed T cell activation and HIV-1 latency reversal did not occur through t...
A CRISPR-engineered bacteriophage T4-COVID vaccine delivers multiple SARS-CoV-2 antigens for pote... more A CRISPR-engineered bacteriophage T4-COVID vaccine delivers multiple SARS-CoV-2 antigens for potential broader protection.
A new bacteriophage T4-induced DNA-dependent ATPase-endonuclease was purified to essential homoge... more A new bacteriophage T4-induced DNA-dependent ATPase-endonuclease was purified to essential homogeneity from an extract of late infected Escherichia coli. Both DNA-dependent ATPase and endonuclease activities co-chromatograph, co-sediment, and have been renatured from a single 43-kilodalton protein eluted following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that both activities are exerted by one multifunctional protein. Duplex, single-stranded, and supercoiled DNAs are all effective activators of the high specific activity ATPase which produces ADP and inorganic PO4. The enzyme displays a broad specificity towards the nucleoside and deoxynucleoside triphosphates, and the ATPase activity is strongly inhibited by DNA-intercalating compounds. The endonuclease appears to be most active on supercoiled DNA, producing double-stranded breaks in duplex DNA, and does not require nucleoside triphosphates. An antiserum against the purified enzyme immunoprecipitated it, inhibited its ATPase activity, and also precipitated from extracts a T4-induced protein of Mr = 43,000. This antigen was not found in uninfected E. coli, or following a gene 55am mutant (late protein synthesis defective) infection, and was not detected following infection with T4 amber mutants of any early capsid protein gene which blocks T4 head protein cleavage in vivo. In a pulse-chase experiment, the radioactive antigen was not found following a pulse of radioactive amino acids, but appeared after a chase with excess nonradioactive amino acids. The enzyme-related antigen is apparently produced by cleavage of a precursor by the T4 head assembly proteinase which processes a number of prohead proteins. These processing reactions are dependent in vivo upon assembly of the prohead and are required for its maturation. The evidence suggests that this enzyme functions in head assembly and DNA packaging, and originates as the cleavage product of a prohead precursor protein.
The objective of this study was to explore the efficacy of low dose of oxcarbazepine (OXC) in adu... more The objective of this study was to explore the efficacy of low dose of oxcarbazepine (OXC) in adult patients with newly diagnosed partial epilepsy in an actual clinical setting. The associated factors influencing the poor control of seizures were also evaluated. The epilepsy database (2010-2014) from the Epilepsy Clinic of West China Hospital was retrospectively reviewed. A total of 102 adult patients with newly diagnosed, previously untreated partial epilepsy initially treated with OXC were included, and divided into good response group (64) and poor response group (38) according to whether they were seizure-free for at least 12 months. There were 27 (26.5%) patients becoming seizure-free with OXC 600mg/day monotherapy. The remaining 75 patients had doses of either increasing OXC to 900mg/day (n=59) or the addition of another antiepileptic drug (AED) (n=16), with another 20 (19.6%) and six (5.9%) patients becoming seizure-free, respectively (P=0.788). In addition, two (2.0%) and ni...
Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuro... more Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuronal cells and efficiently evade the immune system. It has been a major medical challenge to eradicate them and, despite intensive efforts, an effective vaccine is not available. We previously showed that upon infection of antigen-presenting cells, HSV type 1 (HSV-1) rapidly and efficiently downregulates the major histocompatibility complex class I-like antigen-presenting molecule, CD1d, and potently inhibits its recognition by CD1d-restricted natural killer T (NKT) cells. It suppresses CD1d expression primarily by inhibiting its recycling to the cell surface after endocytosis. We identify here the viral glycoprotein B (gB) as the predominant CD1d-interacting protein. gB initiates the interaction with CD1d in the endoplasmic reticulum and stably associates with it throughout CD1d trafficking. However, an additional HSV-1 component, the serine-threonine kinase US3, is required for optimal ...
Studies on human immunodeficiency virus type 1 (HIV-1) diversity are critical for understanding v... more Studies on human immunodeficiency virus type 1 (HIV-1) diversity are critical for understanding viral pathogenesis and the emergence of immune escape variants and for design of vaccine strategies. To investigate HIV-1 population genetics, we used single-genome sequencing to obtain pro-pol and env sequences from longitudinal samples ( n = 93) from 14 acutely or recently infected patients. The first available sample after infection for 12/14 patients revealed HIV-1 populations with low genetic diversity, consistent with transmission or outgrowth of a single variant. In contrast, two patients showed high diversity and coexistence of distinct virus populations in samples collected days after a nonreactive enzyme-linked immunosorbent assay or indeterminate Western blot, consistent with transmission or outgrowth of multiple variants. Comparison of PR and RT sequences from the first sample for all patients with the consensus subgroup B sequence revealed that nearly all nonsynonymous differ...
The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A s... more The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A single structural protein comprises this dodecameric ring substructure of the prohead. Assembly of the phage T4 prohead occurs on the cytoplasmic membrane through a specific attachment at or near the gp20 DNA entrance vertex. An auxiliary head assembly gene product, gp40, was hypothesized to be involved in assembling the gp20 substructure. T4 genes 20,40 and 20 + 40 were cloned into expression vectors under lzpL promoter control. The corresponding T4 gene products were synthesized in high yield and were active as judged by their ability to complement the corresponding infecting T4 mutants in vivo. The cloned T4 gene 20 and gene 40 products were inserted into the cytoplasmic membrane as integral membrane proteins; however, gp20 was inserted into the membrane only when gp40 was also synthesized, whereas gp40 was inserted in the presence or absence of gp20. The gp20 insertion required a membrane potential, was not dependent upon the Escherichia coli groE gene, and assumed a defined membrane-spanning conformation, as judged by specific protease fragments protected by the membrane. The inserted gp20 structure could be probed by antibody binding and protein A-gold immunoelectron microscopy. The dat'a suggest that a specific gp20-gp4Gmembrane insertion structure constitutes the T4 prohead assembly initiation complex.
Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer... more Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer alternatives to whole pathogen vaccines. However, the antigens by themselves are not sufficiently immunogenic and require additives known as adjuvants to enhance immunogenicity and protective efficacy. Assembly of the antigens into virus-like nanoparticles (VLPs) is a better approach as it allows presentation of the epitopes in a more native context. The repetitive, symmetrical, and high density display of antigens on the VLPs mimic pathogen-associated molecular patterns seen on bacteria and viruses. The antigens, thus, might be better presented to stimulate host's innate as well as adaptive immune systems thereby eliciting both humoral and cellular immune responses. Bacteriophages such as phage T4 provide excellent platforms to generate the nanoparticle vaccines. The T4 capsid containing two non-essential outer proteins Soc and Hoc allow high density array of antigen epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Co-delivery of DNAs, targeting molecules, and/or molecular adjuvants provides additional advantages. Recent studies demonstrate that the phage T4 VLPs are highly immunogenic, do not need an adjuvant, and provide complete protection against bacterial and viral pathogens. Thus, phage T4 could potentially be developed as a "universal" VLP platform to design future multivalent vaccines against complex and emerging pathogens.
<p>(<b>A</b>) Schematic of native F1-V, F1mut-V and F1mut-V10. Cyan represents ... more <p>(<b>A</b>) Schematic of native F1-V, F1mut-V and F1mut-V10. Cyan represents the coding sequence of V antigen, and yellow, the putative immunomodulatory sequence that is part of V sequence. Rest of the colors represents the same as described in legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g002" target="_blank">Figure 2</a>. (<b>B</b>) Expression and solubility analysis of F1-V constructs were performed using the B-PER reagent. The samples were analyzed by SDS-PAGE and Coomassie blue staining. The positions of the F1-V protein bands are marked with red arrows. S, soluble fraction (supernatant from 12,000 <i>g</i> centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (<b>C</b>) F1-V, F1mut-V and F1mut-V10 were purified by HisTrap column chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. The calibration graph was generated by passing various molecular weight standards through the same column [Thyroglobulin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), aldolase (158 kDa), Ovalbumin (43 kDa), RNase A (14 kDa), and Albumin (67 kDa)]. The insert shows the purity of F1-V, F1mut-V, and F1mut-V10 proteins following SDS-PAGE and Coomassie blue staining of the peak fractions. The color of arrows corresponds to the color of the elution profiles of various proteins. (<b>D</b>) Stability of F1-V and F1mut-V proteins was tested by treatment with increasing amounts of trypsin at room temperature overnight. The ratios shown above the gel correspond to the ratios of F1-V or F1mut-V proteins to trypsin (wt∶wt). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details.</p
<p>The immunogenicity and protective efficacy of F1mut-V and other plague immunogens were e... more <p>The immunogenicity and protective efficacy of F1mut-V and other plague immunogens were evaluated in a mouse model. (<b>A</b>) Balb/c mice, twelve per group, were vaccinated with various plague antigens adjuvanted with alhydrogel. (<b>B</b>) Immunization scheme. (<b>C</b>) Antigen-specific antibody (IgG) titers were determined by ELISA, using purified V (I), F1mut2 (II), or YscF35/67 (III) as the coating antigen. No significant cross-reactivity was observed between the antibodies produced against one plague antigen versus a different plague antigen that was coated on the ELISA plate. Error bars represent S.D. “***” denotes p<0.001 (ANOVA). (<b>D</b>) Survival of immunized mice against intranasal challenge with 90 LD<sub>50</sub> of <i>Y. pestis</i> CO92. The survived mice were re-challenged with 9,800 LD<sub>50</sub> at day-48 post-first challenge. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.</p
<p>(<b>IgG1) and T<sub>H</sub>2 (IgG2a) responses.</b> The immuniza... more <p>(<b>IgG1) and T<sub>H</sub>2 (IgG2a) responses.</b> The immunization scheme is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g006" target="_blank">Figure 6B</a>. Seven days after the second boost, sera were collected and IgG1 (<b>A</b>) and IgG2a (<b>B</b>) titers were determined by ELISA. F1mut-V was used as the coating antigen, since it covers all the epitopes present in both F1mut-V and F1mut-V10. Note that the sera of the control T4 phage-immunized mice showed higher background. This was because T4 phage, as demonstrated in previous studies, induces a strong antibody response to its components. Consequently, the sera from T4 phage- immunized mice will have increased amounts of IgGs compared to the pre-immune sera, giving more non-specific background at low dilutions of the sera. Data shown are the antibody titers of 12 mice in each group with S.D. (error bars). *, p<0.05; ***, p<0.001 (ANOVA).</p
Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures ... more Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding th...
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