Transplanting Supersites of HIV-1 Vulnerability

<div><p>One strategy for isolating or eliciting antibodies against a specific target region on the envelope glycoprotein trimer (Env) of the human immunodeficiency virus type 1 (HIV-1) involves the creation of site transplants, which present the target region on a heterologous protein scaffold with preserved antibody-binding properties. If the target region is a supersite of HIV-1 vulnerability, recognized by a collection of broadly neutralizing antibodies, this strategy affords the creation of “supersite transplants”, capable of binding (and potentially eliciting) antibodies similar to the template collection of effective antibodies. Here we transplant three supersites of HIV-1 vulnerability, each targeted by effective neutralizing antibodies from multiple donors. To implement our strategy, we chose a single representative antibody against each of the target supersites: antibody 10E8, which recognizes the membrane-proximal external region (MPER) on the HIV-1 gp41 glycoprotein; antibody PG9, which recognizes variable regions one and two (V1V2) on the HIV-1 gp120 glycoprotein; and antibody PGT128 which recognizes a glycopeptide supersite in variable region 3 (glycan V3) on gp120. We used a structural alignment algorithm to identify suitable acceptor proteins, and then designed, expressed, and tested antigenically over 100-supersite transplants in a 96-well microtiter-plate format. The majority of the supersite transplants failed to maintain the antigenic properties of their respective template supersite. However, seven of the glycan V3-supersite transplants exhibited nanomolar affinity to effective neutralizing antibodies from at least three donors and recapitulated the mannose₉-<i>N</i>-linked glycan requirement of the template supersite. The binding of these transplants could be further enhanced by placement into self-assembling nanoparticles. Essential elements of the glycan V3 supersite, embodied by as few as 3 <i>N</i>-linked glycans and ∼25 Env residues, can be segregated into acceptor scaffolds away from the immune-evading capabilities of the rest of HIV-1 Env, thereby providing a means to focus the immune response on the scaffolded supersite.</p></div

Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus

<div><p>Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein—stabilized in the pre-fusion (pre-F) conformation by “DS-Cav1” mutations—elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These “head-only” immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting suggest these head-only RSV F immunogens, engineered to retain the pre-fusion conformation, may have advantages as candidate RSV vaccines.</p></div

Negative-stain electron microscopy of head-only immunogens.

<p>Panels <b>(A-P)</b> show typical 2D averaged classes of i-273,i-693, i-210 and i-447 alone <b>(A, E, I</b> and <b>M)</b>, in complex with D25 (in blue; <b>B, F, J</b> and <b>N</b>), in complex with motavizumab (in green; <b>C, G, K</b> and <b>O</b>) and in complex with both D25 and motavizumab (in blue, green; <b>D, H, L</b> and <b>P</b>). Yellow indicates Fabs of ambiguous identity. Two separate averages are shown for each panel. White scale bars are 50 Å long.</p

Design of RSV F head-only immunogens.

<p><b>(A)</b> Genetic construct of RSV F illustrating the location of the head region. The signal peptide is colored yellow, F₂ is colored blue, F₁ is colored green, the transmembrane region is colored cyan and the cytoplasmic domain is colored gray. <b>(B)</b> Pre-F form of the RSV F trimer. One protomer of RSV F is depicted as a ribbon diagram and colored as in A. The other two protomers are gray surface representations. Antigenic site Ø and site II are red and orange respectively. Domains I, II and III are labeled as defined previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159709#pone.0159709.ref009" target="_blank">9</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159709#pone.0159709.ref035" target="_blank">35</a>]. <b>(C)</b> Models of head-only immunogens i-273, i-693 (upper panels), i-210 and i-447 (lower panels). For each immunogen a cartoon of the genetic construct is depicted (top) and a ribbon diagram (bottom), color-coded as in A and B with brown coloring for purification tags. PDB entries 4JHW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159709#pone.0159709.ref009" target="_blank">9</a>], 1RFO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159709#pone.0159709.ref036" target="_blank">36</a>] and 1GCM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159709#pone.0159709.ref037" target="_blank">37</a>] were used to depict RSV F, the foldon and the coiled coil respectively. RSV F residue numbering follows the numbering in PDB entry 4JHW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159709#pone.0159709.ref009" target="_blank">9</a>].</p

A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

<div><p>Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.</p></div

Head-only RSV F immunogen boost elicits neutralization titers surpassing DS-Cav1.

<p><b>(A)</b> All four immunogens were used to immunize mice two times, resulting in high neutralization titers for i-693 and i-447 above the protective threshold of 100 (dotted line). <i>P</i> values between i-447 and i-210 or i-273 were 0.001 and <0.0001 respectively and those between i-693 and i-210 or i-273 were 0.0014 and <0.0001 respectively. <b>(B)</b> Immunogens i-693 and i-447 were used to boost DS-Cav1 seven weeks later resulting in titers higher than a DS-Cav1 homologous boost. Scatter plots show the geometric mean (numerical value below) with error bars representing the 95% confidence level. <i>P</i> values were determined by two-tailed Mann-Whitney tests. Each group included 10 mice.</p

Characterization of head-only immunogens i-273, i-693, i-210 and i-447.

<p><b>(A-D)</b> Size exclusion chromatography (SEC) analysis of all four immunogens after purification by affinity chromatography. For each immunogen an asterisk indicates the peak that was chosen for antigenic, physical, structural and immunization studies. The calculated molecular weight (MW) in kDa based on elution volume and the predicted MW based on the sequence and predicted glycan content (2.5 kDa/glycan) are also indicated. Immunogens i-273, i-693, i-210 and i-447 were predicted to have two, four, three and three glycans respectively based on the occurrence of NXT/S sequons in the sequence. <b>(E)</b> Reduced SDS-PAGE analysis (3μg/well) of immunogens (1) i-273, (2) i-693, (3) i-210 and (4) i-447 pooled from the asterisk-indicated peaks in (A-D). Doublet bands likely arise from differential glycosylation.</p

Negative stain-electron microscopy of RSV F glycoproteins stabilized by different C-terminal motifs.

<p><b>(A-J)</b> shows typical 2D averaged classes of particles of negatively stained specimens for DS-Cav1 with various disulfide coiled-coil motifs. <b>(A)</b> DS-Cav control; <b>(B)</b> ring A with foldon; <b>(C)</b> ring A without foldon; <b>(D)</b> rings AB with foldon; <b>(E)</b> rings AB without foldon; <b>(F)</b> post-fusion F; <b>(G)</b> rings BCD without foldon; <b>(H)</b> rings ABCD without foldon; <b>(I)</b> rings BCDE without foldon; <b>(J)</b> rings ABCDE without foldon. The scale bar is 10 nm.</p

Boost by head-only RSV F immunogen can focus the RSV-neutralizing response to antigenic sites Ø and II.

<p><b>(A)</b> Recognition of DS-Cav1, DS-Cav1 site Ø knockout (KO) and DS-Cav1 site II KO probes by sera from 2×DS-Cav1 immunized mice boosted with DS-Cav1, i-447 or i-693, respectively or unimmunized mice. <b>(B)</b> Neutralization competition of sera from 2×DS-Cav1 immunized mice boosted with DS-Cav1, i-447 or i-693, respectively. <i>P</i> values for differences between DS-Cav1 and DS-Cav1 site Ø or site II KO probes were all <0.0001. <i>P</i> values were determined by two-tailed Mann-Whitney test.</p

Physical Characterization of Head-only RSV F Immunogens.

<p>Physical Characterization of Head-only RSV F Immunogens.</p