Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10.

UNLABELLED: The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Here, we report the cocrystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to nonneutralizing versions of the 4E10 Fab. Nonneutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed ΔLoop) or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Collectively, the data ruled out major conformational changes of CDR-H3 at any stage during the binding process (equilibrium or transition state). Although these mutations did not impact the affinity of wild-type Fab for the 4E10ep in solution, the two nonneutralizing versions of 4E10 were deficient in binding to MPER inserted in the plasma membrane (mimicking the environment faced by the antibody in vivo). The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear α-helical epitope and likely constrained by the viral membrane lipids. IMPORTANCE: The broadly neutralizing anti-HIV-1 4E10 antibody blocks infection caused by nearly all viral strains and isolates examined thus far. However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1-infected individuals or elicited through vaccination. Impediments to the design of successful 4E10 immunogens are partly attributed to an incomplete understanding of the structural and binding characteristics of this class of antibodies. Since the broadly neutralizing activity of 4E10 is abrogated by mutations of the tip of the CDR-H3, we investigated their impact on binding of the MPER-epitope at the atomic and energetic levels. We conclude that the difference between neutralizing and nonneutralizing antibodies of 4E10 is neither structural nor energetic but is related to the capacity to recognize the HIV-1 gp41 epitope inserted in biological membranes. Our findings strengthen the idea that to elicit similar neutralizing antibodies, the suitable MPER vaccine must be "delivered" in a membrane environment.

A pipeline for facile cloning of antibody Fv domains and their expression, purification, and characterization as recombinant His-tagged IgGs.

We report a functional pipeline for facile conversion of variable Fv domains, typically discovered in antibody discovery programs, into chimeric monoclonal antibodies (mAbs). Often, in initial screenings, a set of candidate mAbs is produced in small volumes and purified from supernatant for testing. Our pipeline also simplifies purification of mAbs by using an extended histidine tag (His-10) fused to the C-terminus of the light chain. Both the length of the His-10 and its location have been shown to affect the efficacy of mAb purification using an inexpensive nickel-based resin at neutral pH. Our antibody cloning and purification pipeline, when followed together with detection and affinity measurements, can be smoothly incorporated into an antibody discovery workflow.

Neutralizing epitopes in the membrane-proximal external region of HIV-1 gp41 are influenced by the transmembrane domain and the plasma membrane.

Failure to elicit broadly neutralizing (bNt) antibodies (Abs) against the membrane-proximal external region of HIV-1 gp41 (MPER) reflects the difficulty of mimicking its neutralization-competent structure (NCS). Here, we analyzed MPER antigenicity in the context of the plasma membrane and identified a role for the gp41 transmembrane domain (TM) in exposing the epitopes of three bNt monoclonal Abs (MAbs) (2F5, 4E10, and Z13e1). We transiently expressed DNA constructs encoding gp41 ectodomain fragments fused to either the TM of the platelet-derived growth factor receptor (PDGFR) or the gp41 TM and cytoplasmic tail domain (CT). Constructs encoding the MPER tethered to the gp41 TM followed by a 27-residue CT fragment (MPER-TM1) produced optimal MAb binding. Critical binding residues for the three Nt MAbs were identified using a panel of 24 MPER-TM1 mutants bearing single amino acid substitutions in the MPER; many were previously shown to affect MAb-mediated viral neutralization. Moreover, non-Nt mutants of MAbs 2F5 and 4E10 exhibited a reduction in binding to MPER-TM1 and yet maintained binding to synthetic MPER peptides, indicating that MPER-TM1 better approximates the MPER NCS than peptides. Replacement of the gp41 TM and CT of MPER-TM1 with the PDGFR TM reduced binding by MAb 4E10, but not 2F5, indicating that the gp41 TM plays a pivotal role in orienting the 4E10 epitope, and more globally, in affecting MPER exposure.

Silicon supplementation improves early blight resistance in Lycopersicon esculentum Mill. by modulating the expression of defense-related genes and antioxidant enzymes.

Early blight is the most devastating disease in tomato which causes huge yield losses across the globe. Hence, development of specific, efficient and ecofriendly tools are required to increase the disease resistance in tomato plants. Here, we systematically investigate the defensive role and priming effect of silicon (Si) in tomato plants under control and infected conditions. Based on the results, Si-treated tomato plants showed improved resistance to Alternaria solani as there was delay in symptoms and reduced disease severity than non-Si-treated plants. To further examine the Si-mediated molecular priming in tomato plants, expression profiling of defense-related genes like PR1, PR2, WRKYII, PR3, LOXD and JERF3 was studied in control, Si-supplemented, A. solani-inoculated and Si + A. solani-inoculated plants. Interestingly, Si significantly increased the expression of jasmonic acid (JA) marker genes (PR3, LOXD and JERF3) than salicylic acid (SA) marker genes (PR1, PR2 and WRKYII). However, Si + A. solani-inoculated plants showed higher expression levels of defence genes except WRKYII than A. solani-inoculated or Si-treated plants. Furthermore, pre-supplementation of Si to A. solani-infected tomato plants showed increased activity of antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase (POD) than control, Si-treated and A. solani-inoculated plants. Altogether, present study highlights the defensive role of Si in tomato plants in response to A. solani by increasing not only the transcript levels of defense signature genes, but also the activity of antioxidant enzymes.

Structural basis for broad neutralization of HIV-1 through the molecular recognition of 10E8 helical epitope at the membrane interface.

The mechanism by which the HIV-1 MPER epitope is recognized by the potent neutralizing antibody 10E8 at membrane interfaces remains poorly understood. To solve this problem, we have optimized a 10E8 peptide epitope and analyzed the structure and binding activities of the antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction (MPER-N-TMD; Env residues 671-687). The MPER-N-TMD helix projects beyond the tip of the heavy-chain complementarity determining region 3 loop, indicating that the antibody sits parallel to the plane of the membrane in binding the native epitope. Biophysical, biochemical and mutational analyses demonstrated that strengthening the affinity of 10E8 for the TMD helix in a membrane environment, correlated with its neutralizing potency. Our research clarifies the molecular mechanisms underlying broad neutralization of HIV-1 by 10E8, and the structure of its natural epitope. The conclusions of our research will guide future vaccine-design strategies targeting MPER.

CD8+ T-cells: function and response to HIV infection.

CD8+ T-cells are a critical component of the cellular immune response and they play an important role in the control of viral infection. During HIV infection, CD8+ T-cells are able to recognize infected cells through an MHC-I dependent process and are able to lyse cells harboring viral infection by the secretion of perforin and granzymes. These cytotoxic T-lymphocytes (CTL) can also eliminate virally infected cells through the engagement of death-inducing ligands expressed by CD8+ T-cells with death receptors on the surface of the infected cell. In addition, CD8+ CTL secrete soluble factors such as beta-chemokines and the CD8+ antiviral factor (CAF) that suppress viral binding and transcription, respectively. In order for HIV to survive the pressures placed upon it by the immune system, the virus has adopted numerous strategies to evade the CD8+ T-cell response. The high mutation rate of HIV has allowed the virus to escape CD8+ T-cell recognition in addition to its ability to down-regulate surface MHC-I expression from infected cells. Also, by altering the pattern of cytokine production and engagement of cellular receptors, HIV disrupts proper CD8+ T-cell signaling. The resultant improper T-cell receptor (TcR) stimulation creates an anergic state in these cells. By affecting the function of CD4+ T-cells and antigen presenting cells that are required for proper CD8+ T-cell maturation, HIV is able to decrease the circulating pool of effector and memory CD8+ T-cells that are able to combat viral infection. The end result is the aberration of CD8+ T-cell function.

Human immunodeficiency virus-1 infection protects against a Tc1-to-Tc2 shift in CD8(+) T cells.

Despite the reports of dysfunction of the lytic abilities of CD8(+) T cells during human immunodeficiency virus-1 (HIV-1) disease progression, the effects of infection on the noncytolytic functions of CD8(+) T cells have not been well characterized to date. We examined the effect of HIV-1 infection on the cytokine and chemokine responses of peripheral blood-derived CD8(+) T cells in an in vitro system. Activation of HIV-1-infected CD8(+) T cells with phytohemagglutinin resulted in a 4- to 8-fold increase in the production of macrophage inflammatory protein (MIP)-1α, MIP-1ß, regulated on activation normal T-cell expressed and secreted, and interleukin (IL)-16. Treatment of activated HIV-1-infected CD8(+) T cells with anti-CD3 monoclonal (M) antibody (Ab) and IL-15 induced strong production of interferon-γ (IFN-γ). Treatment of cells with anti-IL-12 MAb and IL-4 to induce a Tc1-to-Tc2 shift resulted in no change in viral production levels or IFN-γ production within the HIV-1-infected CD8(+) T cell population. Initiation of a Tc2-to-Tc1 shift resulted in a 6-fold increase in HIV-1 replication and 2- to 3-fold higher levels of IFN-γ, demonstrating that infection can protect against a Tc1-to-Tc2 shift in CD8(+) T cells.

Infection of CD8+CD45RO+ memory T-cells by HIV-1 and their proliferative response.

CD8+ T-cells are involved in controlling HIV-1 infection by eliminating infected cells and secreting soluble factors that inhibit viral replication. To investigate the mechanism and significance of infection of CD8+ T-cells by HIV-1 in vitro, we examined the susceptibility of these cells and their subsets to infection. CD8+ T-cells supported greater levels of replication with T-cell tropic strains of HIV-1, though viral production was lower than that observed in CD4+ T-cells. CD8+ T-cell infection was found to be productive through ELISA, RT-PCR and flow cytometric analyses. In addition, the CD8+CD45RO+ memory T-cell population supported higher levels of HIV-1 replication than CD8+CD45RA+ naïve T-cells. However, infection of CD8+CD45RO+ T-cells did not affect their proliferative response to the majority of mitogens tested. We conclude, with numerous lines of evidence detecting and measuring infection of CD8+ T-cells and their subsets, that this cellular target and potential reservoir may be central to HIV-1 pathogenesis.

Proportion of HIV-1 infected CD8+CD4- T lymphocytes in vivo.

The proportion and significance of HIV-1 infection of CD8+ T-cells was examined in a patient cohort of HIV-1 seropositive (n=28) and seronegative individuals (n=4). It was hypothesized that irrespective of the clinical status of the patients, productively HIV-1 infected CD8+ T-cells would be found and these cells would contribute to the plasma viral load in vivo. Flow cytometric analysis using fluorochrome-conjugated antibodies, RT-PCR analysis using HIV-1(pol) specific primers, and quantification of HIV-1 viral transcripts by ex vivo culture of isolated CD8+ T-cells were employed. In 22 of the 28 patient samples analyzed, a significantly higher proportion of cells with expression of CD8+HIV-1(gag)+ than of CD4+HIV-1(gag)+ T-cells was observed (36.9% +/- 10.0% vs 26.4% +/- 13.1% respectively, p< 0.01). No correlation was observed between absolute CD4 counts, CD8 counts, plasma viral load and CD8+ T cell infection. RT-PCR analysis indicated the presence of HIV-1 transcripts in the ex vivo isolated CD8+ T-cell population. Ex vivo isolated CD8+ T-cells demonstrated productive infection over time. We conclude, with three lines of evidence detecting and measuring HIV-1 infection of CD8+ T-lymphocytes, that this cellular target and reservoir may be central to HIV-1 pathogenesis.

Binding to decay-accelerating factor is not required for infection of human leukocyte cell lines by enterovirus 70.

Enterovirus 70 (EV70) is one of several human enteroviruses that exhibit a propensity for infecting the central nervous system (CNS). The mechanisms by which neurotropic enteroviruses gain access to and invade the CNS are poorly understood. One possibility is that circulating leukocytes become infected and carry neurotropic enteroviruses to the CNS. We examined the ability of EV70 to infect cell lines derived from lymphoid, myeloid, and monocytic lineages. Most leukocyte cell lines tested bound radiolabeled EV70 and were permissive for EV70 replication, suggesting that EV70, in contrast to other enteroviruses, has an in vitro tropism that includes lymphoid, monocytic, and myeloid cell lines. For some of the cell lines, virus binding and infection correlated with surface expression of decay-accelerating factor (DAF), an attachment protein for EV70 on HeLa cells. However, EV70 also adsorbed to and infected cell lines that expressed little or no DAF. In contrast to what was observed for HeLa cells, neither DAF-specific monoclonal antibodies nor phosphatidylinositol-specific phospholipase C treatment inhibited EV70 binding to permissive leukocyte cell lines, and antibody blockade of DAF had little or no effect on EV70 replication. We also found that neither the human coxsackievirus-adenovirus receptor nor intercellular cell adhesion molecule 1, which mediate the entry of coxsackie B viruses and coxsackievirus A21, respectively, functions as a receptor for EV70. EV70 binding to all cell lines was sensitive to sialidase treatment and to inhibition of O glycosylation by benzyl N-acetyl-alpha-D-galactosaminide. Taken together, these results suggest that a sialylated molecule(s) other than DAF serves as a receptor for EV70 on permissive human leukocyte cell lines.