Oligomeric and conformational properties of a proteolytically mature,disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein

We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1(JR-FL) SOS gp140, proteolytically uncleaved gp140 (gp140(UNC)), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140(UNC), SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140(UNC) displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41(ECTO)) occludes the "nonneutralizing" face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140(UNC) comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140(UNC) were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140(UNC)) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.     

The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5

The human monoclonal antibody 2F5 neutralizes primary human immunodeficiency virus type 1 (HIV-1) with rare breadth and potency. A crystal structure of a complex of 2F5 and a peptide corresponding to its core epitope on gp41, ELDKWAS, revealed that the peptide interacts with residues at the base of the unusually long (22-residue) third complementarity-determining region of the heavy chain (CDR H3) but not the apex. Here, we perform alanine-scanning mutagenesis across CDR H3 and make additional substitutions of selected residues to map the paratope of Fab 2F5. Substitution of residues from the base of the H3 loop or from CDRs H1, H2, and L3, which are proximal to the peptide, significantly diminished the affinity of Fab 2F5 for gp41 and a short peptide containing the 2F5 core motif. However, nonconservative substitutions to a phenylalanine residue at the apex of the H3 loop also markedly decreased 2F5 binding to both gp41 and the peptide, suggesting that recognition of the core epitope is crucially dependent on features at the apex of the H3 loop. Furthermore, substitution at the apex of the H3 loop had an even more pronounced effect on the neutralizing activity of 2F5 against three sensitive HIV-1. These observations present a challenge to vaccine strategies based on peptide mimics of the linear epitope.     

Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies

Virion capture assays, in which immobilized antibodies (Abs) capture virus particles, have been used to suggest that nonneutralizing Abs bind effectively to human immunodeficiency virus type 1 (HIV-1) primary viruses. Here, we show that virion capture assays, under conditions commonly reported in the literature, give a poor indication of epitope expression on the surface of infectious primary HIV-1. First, estimation of primary HIV-1 capture by p24 measurements shows a very poor correlation with an estimation based on infectivity measurements. Second, virion capture appears to require relatively low Ab affinity for the virion, as shown by the ability of a monoclonal Ab to capture a wild-type and a neutralization escape variant virus equally well. Nevertheless, in a more interpretable competition format, it is shown that nonneutralizing anti-CD4 binding site (CD4bs) Abs compete with a neutralizing anti-CD4bs Ab (b12) for virus capture, suggesting that the nonneutralizing anti-CD4bs Abs are able to bind to the envelope species that is involved in virion capture in these experiments. However, the nonneutralizing anti-CD4bs Abs do not inhibit neutralization by b12 even at considerable excess. This suggests that the nonneutralizing Abs are unable to bind effectively to the envelope species required for virus infectivity. The results were obtained for three different primary virus envelopes. The explanation that we favor is that infectious HIV-1 primary virions can express two forms of gp120, an accessible nonfunctional form and a functional form with limited access. Binding to the nonfunctional form, which needs only to be present at relatively low density on the virion, permits capture but does not lead to neutralization. The expression of a nonfunctional but accessible form of gp120 on virions may contribute to the general failure of HIV-1 infection to elicit cross-neutralizing Abs and may represent a significant problem for vaccines based on viruses or virus-like particles.     

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.     

Mitochondrial control-region sequences in two shorebird species, the turnstone and the dunlin, and their utility in population genetic studies

We determined the mitochondrial control-region sequences of five turnstones (Arenaria interpres) and three dunlins (Calidris alpina). Comparisons revealed that the central part (part II) is conserved relative to much more variable parts at the beginning (part I) and the end (part III). This pattern of sequence conservation is also found in the control regions of other vertebrates. The average sequence divergence between turnstone and dunlin was 21.8% for part I, 7.5% for part II, and 29.5% for part III. Within-species sequence divergence over the entire control region was much lower, at 0.9% for turnstones and 2.0% for dunlins. In both shorebird species, part III contains a repetitive sequence composed only of A and C nucleotides, which has not been found in the control regions of other birds. A survey of the part I sequences of 25 turnstones and 25 dunlins sampled around the world revealed that these species have very different population genetic structures. Dunlins are not only much more differentiated in their sequences but also have a strongly subdivided population genetic structure. Pleistocene vicariant events combined with strong natal philopatry and high mutation rates of the sequences are likely responsible for this population genetic subdivision. Conversely, part I sequences of turnstones are weakly differentiated and are geographically unstructured. We argue that this is not the result of global gene flow but that, instead turnstones have recently expanded from a refugial population that was bottlenecked.      

'Navius' kissing stents for coronary bifurcation stenosis,recreating a new metallic carina: an IVUS-assessed case report

We report a case of implantation of a new design of stent which allows creation of a double-hemispheric lumen for the treatment of a bifurcational stenosis. The unfavourable outcome following the implantation of this stent is described.     

Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies. Identification of a functional polymorphism to human IgG2 anti-CD3.

T cell activation induced by mouse anti-CD3 mAb has shown to be dependent on the Ig isotype of these antibodies. A study of isotype dependency of human antibodies, however, seems more relevant to human effector systems, especially in view of the availability of humanized antibodies for clinical applications. We constructed a panel of mouse and mouse/human chimeric anti-CD3 mAb, which differ only in their CH region and hence have identical binding sites and affinity. By using these antibodies, we now studied their ability to induce T cell proliferation in human PBMC and analyzed the classes of IgG FcR involved in these responses. The human (h)IgG1, hIgG3, and hIgG4, as well as mouse (m)IgG2a and mIgG3 anti-CD3 mAb induced an Fc gamma RI (CD64)-dependent T cell proliferation in all donors. Activation with hIgG2 and mIgG1 anti-CD3 mAb was observed to be mediated via the low affinity Fc gamma RII (CD32). It was found that leukocytes in a normal donor population display a functional polymorphism with respect to hIgG2 anti-CD3 responsiveness. This polymorphism was found to be inversely related to the previously defined Fc gamma RII-polymorphism to mIgG1 anti-CD3 mAb. Monocytes expressing the Fc gamma RII mIgG1 low responder (LR) allele support hIgG2 anti-CD3 induced T cell proliferation efficiently, whereas cells homozygous for the Fc gamma RII mIgG1 high responder (HR) allele do not. This observation could be confirmed in T cell activation studies using hFc gamma RIIa-transfected mouse fibroblasts, expressing either the mIgG1 anti-CD3 HR or LR Fc gamma RII-encoding cDNA.     

Effect of drift sampler exposure time and net mesh size on invertebrate drift density in the Njoro River,Kenya

Although invertebrate drift is an important ecological process in lotic ecosystems, very little is known about it in Kenyan rivers. The primary aim of this study was to investigate the effect of driftnet mesh size and exposure duration on drift density in 2017. Drift samples were dominated by Chironomidae, Baetidae, Simuliidae, Caenidae and Culicidae. The 100 µm mesh driftnet had the highest mean invertebrate density, followed by the 250 µm and 500 µm nets. Invertebrate drift densities decreased with increased exposure time. This study demonstrates that sampler mesh size and exposure time should be taken into account when characterising invertebrate drift in streams. Future studies should consider sampling different biotopes and during different seasons.     

Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off.

IgG1b12, a human monoclonal antibody (MAb) to an epitope overlapping the CD4-binding site on gp120, has broad and potent neutralizing activity against most primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess whether and how escape mutants resistant to IgG1b12 can be generated, we cultured primary HIV-1 strain JRCSF in its presence. An escape mutant emerged which was approximately 100-fold more resistant to neutralization by IgG1b12. Both virion-associated and solubilized gp120 from this variant had a reduced affinity for IgG1b12, and sequencing of its env gene showed that amino acid substitutions had occurred at three positions within gp120. Two (D164N and D182N) were located in V2, and one (P365L) was in C3. By site-directed mutagenesis, we demonstrated that the D182N and P365L mutations, but not D164N, contribute to the IgG1b12-resistant phenotype. However, the former two substitutions, individually or in combination, hinder the replication of the neutralization-resistant virus. Introduction of the D164N substitution into the P365L variant results in a nonviable virus (D164N/P365L). In contrast, addition of D164N to the D182N or D182N/P365L mutant partially restored replicative function to near wild-type levels. Furthermore, we found that all of the IgG1b12-resistant mutant viruses remained sensitive to other human MAbs, such as 2G12 and 2F5, and to the CD4-IgG molecule, except that the P365L-containing mutant was slightly resistant to CD4-IgG. These results suggest that escape from IgG1b12 neutralization is due to a local rather than a global modification of the gp120 structure. Our findings have implications for the therapeutic and prophylactic applications of antibodies for HIV-1 infection.     

The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.