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  • The American Society for Microbiology (ASM)  (14)
  • Blackwell Publishing Ltd  (9)
  • American Association for the Advancement of Science (AAAS)  (1)
  • 11
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    A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization [Genome Replication and Regulation of Viral Gene Expression] (2012)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2012-09-21
    Description: Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S -transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro . We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [ 35 S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 12
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    Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin [Vaccines and Antiviral Agents] (2012)
    The American Society for Microbiology (ASM)
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    Publication Date: 2012-10-07
    Description: Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA 3 ) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA 3 glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5 3 ) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5 3 preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man 9 GlcNAc 2 moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man 5 GlcNAc 2 moieties derived from HEK293S GnTI(–) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(–) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N -acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N -acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.
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    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 13
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    The microanatomic segregation of selection by apoptosis in the germinal center (2017)
    American Association for the Advancement of Science (AAAS)
    In: Science
    Details
    Publication Date: 2017-10-13
    Description: B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.
    Keywords: Immunology, Online Only
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Geosciences , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 14
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    Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes [Vaccines and Antiviral Agents] (2017)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2017-05-27
    Description: Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 15
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    HIV-1 Escape from a Peptidic Anchor Inhibitor through Stabilization of the Envelope Glycoprotein Spike [Vaccines and Antiviral Agents] (2016)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2016-11-15
    Description: The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells by using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be understood better in order to optimally design vaccines and therapeutics. We generated viruses with resistance to VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K, and H66R) that rendered these viruses dependent on the inhibitor. These viruses could infect target cells only when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors and provide new ways to stabilize Env trimer vaccines. IMPORTANCE Because of the rapid development of HIV-1 drug resistance, new drug targets need to be explored continuously. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccines.
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    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 16
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    Reducing V3 Antigenicity and Immunogenicity on Soluble, Native-Like HIV-1 Env SOSIP Trimers [Vaccines and Antiviral Agents] (2017)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2017-07-13
    Description: Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such "off-target" immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N -glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man 6 GlcNAc 2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged. IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.
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    Topics: Medicine
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  • 17
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    High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers [Vaccines and Antiviral Agents] (2017)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2017-10-28
    Description: Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies. IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.
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    Topics: Medicine
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  • 18
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    Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers [Vaccines and Antiviral Agents] (2015)
    The American Society for Microbiology (ASM)
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    Publication Date: 2015-11-06
    Description: We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140 UNC -Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41 ECTO ) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41 ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ~20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140 UNC -Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. IMPORTANCE Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.
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    Topics: Medicine
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  • 19
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    Chemical Cross-Linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens [Vaccines and Antiviral Agents] (2015)
    The American Society for Microbiology (ASM)
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    Publication Date: 2015-12-31
    Description: Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens. IMPORTANCE The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.
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    Topics: Medicine
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  • 20
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    Structural Characterization of Cleaved, Soluble HIV-1 Envelope Glycoprotein Trimers [Structure and Assembly] (2013)
    The American Society for Microbiology (ASM)
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    Publication Date: 2013-08-14
    Description: Human immunodeficiency virus type 1 (HIV-1) infection is a significant global public health problem for which development of an effective prophylactic vaccine remains a high scientific priority. Many concepts for a vaccine are focused on induction of appropriate titers of broadly neutralizing antibodies (bNAbs) against the viral envelope (Env) glycoproteins gp120 and gp41, but no immunogen has yet accomplished this goal in animals or humans. One approach to induction of bNAbs is to design soluble, trimeric mimics of the native viral Env trimer. Here, we describe structural studies by negative-stain electron microscopy of several variants of soluble Env trimers based on the KNH1144 subtype A sequence. These Env trimers are fully cleaved between the gp120 and gp41 components and stabilized by specific amino acid substitutions. We also illustrate the structural consequences of deletion of the V1/V2 and V3 variable loops from gp120 and the membrane-proximal external region (MPER) from gp41. All of these variants adopt a trimeric configuration that appropriately mimics native Env spikes, including the CD4 receptor-binding site and the epitope for the VRC PG04 bNAb. These cleaved, soluble trimer designs can be adapted for use with multiple different env genes for both vaccine and structural studies.
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    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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