Making Diagnostic Testing for Lyme Disease More Approachable

The CDC’s recommended testing and treatment guidelines can be confusing for patients and healthcare providers alike. There is often a knowledge gap in understanding what is being analyzed in serologic tests as well as why two step testing is employed. With the increasing incidence of Lyme in the northeast it is as important as ever to ensure that there is an approachable resource for patients to learn from. Better patient care can be provided while also reducing unnecessary testing and resource overutilization by empowering patients with a better understanding of the process of diagnosis of Lyme diseasehttps://scholarworks.uvm.edu/fmclerk/1285/thumbnail.jp

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp) 140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120-gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120-gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41(ECTO)) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41(ECTO). Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41(ECTO) epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluate

RESEARCH Open Access

structural and vaccine studie

A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene

Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies. The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccin

A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies

A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogen

BG505 SOSIP.664 gp140 antigenicity by SPR.

<p>BG505 SOSIP.664-His gp140 trimers were immobilized on NTA chips (<b>A</b>–<b>D</b>). The sensorgrams show the response (RU) over time (s) using IgGs at 1,000 nM (150,000 ng/ml) (A–C) or Fabs at 500 nM (25,000 ng/ml) (D). The association phase was 300 s and dissociation was followed over 600 s. (<b>A</b>) 2G12 (high), PGT135 (intermediate), PGV04 (high), b6 (marginal), b12 (undetectable) and F240 (undetectable); (<b>B</b>) PG9, PG16, PGT145 (all intermediate); (<b>C</b>) PGT121 (intermediate), PGT123 (high), PGT128 (high), 14e (low); (<b>D</b>) Fabs of PGV04 (intermediate), b6, b12, and F240 (undetectable). (<b>E</b>) In an alternative approach, Env-reactive MAb was captured by anti-Fc Ab on the chip and the responses to BG505 SOSIP.664 gp140 trimers (200 nM; 78,000 ng/ml) were followed: 2G12 (high), PGT128 (high), b6 (low), b12, F240 (undetectable). Each curve represents one of 2–3 similar replicates.</p

Design and biochemical characterization of BG505 SOSIP.664 gp140 trimers.

<p>(<b>A</b>) Linear representation of the BG505 gp160, SOSIP.664 gp140, SOSIP.664-D7324 gp140 and gp120-D7324 Env proteins. Modifications compared to the original BG505 gp160 sequence are indicated in red and mentioned in the text. The following changes were made to the wild type BG505 amino acid sequence: 1) The tissue plasminogen activator (tPA) signal peptide replaced the natural one; 2) the gp41 transmembrane (TM) and cytoplasmic tail (CT) domains were deleted to create a soluble gp140; 3) the A501C and T605C substitutions were made to form a disulfide bond between gp120 and gp41_ECTO<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Binley1" target="_blank">[5]</a>; 4) the I559P substitution was included to promote trimerization <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Sanders1" target="_blank">[6]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Sanders4" target="_blank">[80]</a>; 5) an optimal cleavage site (RRRRRR; R6) replaces the natural one, REKR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Binley2" target="_blank">[31]</a>; 6) truncation of the MPER from residue-664 prevents aggregation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Khayat1" target="_blank">[23]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Klasse1" target="_blank">[24]</a>; 7) the T332N substitution facilitates binding of bNAbs dependent on glycan-N332. The D7324- and His-tags are indicated in yellow. Env sub-domains are indicated: 5 conserved domains (C1–C5); 5 variable domains (V1–V5); heptad repeats 1 and 2 (HR1, HR2); the membrane proximal external region (MPER); the transmembrane domain (TM); and the cytoplasmic tail (CT). The glycan assignments in Env are based on previous studies using gp120 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Cutalo1" target="_blank">[81]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Leonard1" target="_blank">[82]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Zhu1" target="_blank">[83]</a>, but may be different for trimeric Env <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Bonomelli1" target="_blank">[84]</a>. The amino acid sequence of BG505 SOSIP.664 is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618.s001" target="_blank">Fig. S1</a>. (<b>B</b>) SEC profile of 2G12-purified BG505 SOSIP.664 gp140 expressed in CHO-K1 cells. A Superdex 200 26/60 column was used. (<b>C</b>) Analytical SEC profile of 2G12/SEC-purified BG505 SOSIP.664 trimer re-run on a Superose 6 10/30 column. (<b>D</b>) BN-PAGE analysis of CHO-K1 expressed, 2G12-purified BG505 SOSIP.664 gp140, stained by Coomassie blue. The m.w. of marker (M) proteins (thyroglobulin and ferritin) are indicated. (<b>E</b>) BN-PAGE analysis of 2G12/SEC-purified BG505 SOSIP.664 gp140, stained by Coomassie blue. (<b>F</b>) SDS-PAGE analysis using a 4–12% Bis-Tris Nu-PAGE gel of 2G12/SEC-purified BG505 SOSIP.664 gp140, under non-reducing and reducing conditions, followed by Coomassie blue staining. (<b>G</b>) SDS-PAGE analysis using a 10% Tris-Glycine gel of 2G12/SEC-purified BG505 SOSIP.664 gp140, under non-reducing and reducing conditions, followed by silver staining. The conversion of the gp140 band to gp120 and the appearance of a gp41_ECTO band under reducing conditions is indicative of cleavage.</p

Binding of non-NAbs to BG505 SOSIP.664 gp140 trimers as observed by negative stain EM.^a

a<p>Representative class averages are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat-1003618-g010" target="_blank">Fig. 10</a>.</p

Thermodynamic parameters of PGT121, PGT128, 2G12 and PG9 binding to BG505 SOSIP.664 gp140 trimers measured by isothermal titration calorimetry.

a<p>The reported values are averages from at least two independent measurements. The associated errors are approximately 10% of the average. Representative isotherms are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat-1003618-g008" target="_blank">Fig. 8</a>.</p>b<p>The change in Gibbs free energy (ΔG) was determined using the relationship: ΔG_binding = RTlnK_d<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-deAzevedo1" target="_blank">[87]</a>.</p>c<p>The stoichiometry of binding (N) is directly affected by errors in protein concentration measurements, sample impurity and heterogeneity of gp140 glycans.</p>d<p>Dissociation constant associated with a second (low affinity) binding event.</p>e<p>The binding isotherms do not allow the stoichiometry and enthalpy associated with the second binding event to be determined accurately.</p>f<p>Data previously described elsewhere <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003618#ppat.1003618-Julien2" target="_blank">[27]</a>.</p