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. 2023 Sep 22;19(9):e1011584.
doi: 10.1371/journal.ppat.1011584. eCollection 2023 Sep.

Structure-based design of a single-chain triple-disulfide-stabilized fusion-glycoprotein trimer that elicits high-titer neutralizing responses against human metapneumovirus

Li Ou  1 , Steven J Chen  1 , I-Ting Teng  1 , Lijuan Yang  2 , Baoshan Zhang  1 , Tongqing Zhou  1 , Andrea Biju  1 , Cheng Cheng  1 , Wing-Pui Kong  1 , Nicholas C Morano  3   4 , Erik-Stephane D Stancofski  1 , John-Paul Todd  1 , Yaroslav Tsybovsky  5 , Shuishu Wang  1 , Cheng-Yan Zheng  1 , John R Mascola  1 , Lawrence Shapiro  3   4 , Ruth A Woodward  1 , Ursula J Buchholz  2 , Peter D Kwong  1
Affiliations

Structure-based design of a single-chain triple-disulfide-stabilized fusion-glycoprotein trimer that elicits high-titer neutralizing responses against human metapneumovirus

Li Ou et al. PLoS Pathog. .

Abstract

The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens.

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Conflict of interest statement

LO, BZ, TZ, WPK, YT, JRM, UJB, and PDK are inventors on a U.S. Patent Application filed on their behalf by the National Institutes of Health. The other authors declare no competing interests.

Figures

Fig 1
Fig 1. Modification of F2-F1 linkage further stabilize HMPV F trimer in a prefusion state.
(A) Structure-based design of a single chain pre-F HMPV F based on PDB ID 5WB0; additional designs are shown in S1 Table; D is length change compared to the v3B. (B) SDS-PAGE analysis of interprotomer disulfide-stabilized HMPV F prefusion variants; (C) Analysis of single chain HMPV F prefusion variants by size exclusion chromatography with yields (top row) and immunogen binding affinity to antibodies (bottom row); (D) Negative-stain electron micrographs of v3B single chain variants. 2D classes indicate that most trimers are in prefusion conformation which lack the long tail (pseudo-colored in cyan), whereas some F trimers are in postfusion conformation which appear as elongated torpedo shape with a tail (pseudo-colored in red). Scale bar = 200 Å.
Fig 2
Fig 2. Additional disulfide-bonds can stabilize HMPV F trimer in a prefusion state.
(A) Structure-based design of interprotomer disulfides based on the prefusion structure of HMPV F (PDB ID 5WB0); additional designs are shown in S1 Table. (B) Properties of HMPV Fs. SDS-PAGE analysis of interprotomer disulfide-stabilized HMPV F prefusion variants. (C) Analysis of HMPV F prefusion variants by size exclusion chromatography SEC and expression level (top row) and binding affinity (bottom row) of HMPV designs with additional disulfide bonds. (D) Negative-stain electron micrographs of HMPV F trimer variants. 2D classes indicate the F proteins are in prefusion conformation. Scale bar = 200 Å.
Fig 3
Fig 3. Pro mutations can increase yield and enhance recognition by the prefusion specific antibody, MPE8.
(A) Structure-based design of proline substitutions based on prefusion-postfusion conformation change structure of HMPV F (PDB ID 5WB0) (B) Analysis interprotomer disulfide-stabilized HMPV F prefusion variants by size exclusion chromatography (C) SDS-PAGE analysis of interprotomer disulfide-stabilized HMPV F prefusion variants. (D) Additional proline substitutions increase the expression yield of HMPV F, the yield of v3B_D12 is show as dotted line. (E) The binding affinity of HMPV designs to MPE8, the affinity of v3B_D12 is show as dotted line.
Fig 4
Fig 4. Cryo-EM structure of prefusion-stabilized HMPV F with MPE8-variable domain (Fv) provides details of prefusion structure and of disulfide bonds formation.
(A) Structure of HMPV F trimer (v3B Δ12_D454C-V458C) with single chain MPE8 Fv. Cryo-EM density map and fitted coordinates of HMPV F protomers were colored in cyan, violet and lime, respectively. The density of the scFV of MPE8 was colored in orange (left), while the VH and VL coordinates were colored orange and slate (right). (B) Cryo-EM structure confirms design of intra-chain disulfide bonds and linker between F1 and F2. Each design and its observed EM density map were shown in zoom-in boxes. Density maps were contoured at 1.5 to 4σ in gray meshes. Even though the density for the interchain disulfide bond between Cys84 and Cys249 was not clear, SDS-PAGE indicated single chain trimeric F converted monomeric form in the presence of reducing agent DTT, confirming the formation of interchain disulfide bond (right). Molecular weight marker was run alongside the HMPV F samples. (C) Interactions between HMPV F and MPE8. The epitope of MPE8 located at the interface between two HMPV F protomers is colored orange (top), CDR H3 of MPE8 reaches into the shallow depression at the protomer junction.
Fig 5
Fig 5. Prefusion-stabilized HMPV F variants induce high titer neutralizing responses in mice.
(A) Immunization regimen for CB6F1J mice (n = 10 /group) with two HMPV F immunizations followed by serum analysis at week 5. (B) Elicited HMPV F neutralization by prefusion F immunogens. Limit of detection (LOD) is shown as an arrow.
Fig 6
Fig 6. Immunization of rhesus macaques shows interprotomer disulfide-stabilized variants of either prefusion or postfusion HMPV F induce neutralizing responses many times the average titer in healthy adult humans.
(A) Immunization regimen for rhesus (n = 5/group) with two HMPV F immunizations followed by serum analysis at week 6. (B) Neutralizing responses graphed with geometric mean titers provided. Limit of detection is shown as a dotted line.
Fig 7
Fig 7. Human globulin analysis indicates prefusion conformation stabilized F glycoprotein absorbs neutralizing responses more efficiently than the postfusion form.
(A) Experimental scheme for the depletion of Flebogamma. (B) Depletion of Flebogamma with prefusion- and postfusion-stabilized HMPV(top panel), RSV (middle panel), and PIV3 F (bottom panel) glycoproteins.
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References

    1. Collins PL, Melero JA. Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus Res. 2011;162(1–2):80–99. Epub 20110922. doi: 10.1016/j.virusres.2011.09.020 ; PubMed Central PMCID: PMC3221877. - DOI - PMC - PubMed
    1. Rima B, Collins P, Easton A, Fouchier R, Kurath G, Lamb RA, et al.. ICTV Virus Taxonomy Profile: Pneumoviridae. J Gen Virol. 2017;98(12):2912–3. Epub 20171031. doi: 10.1099/jgv.0.000959 ; PubMed Central PMCID: PMC5775899. - DOI - PMC - PubMed
    1. Rubin S, Eckhaus M, Rennick LJ, Bamford CG, Duprex WP. Molecular biology, pathogenesis and pathology of mumps virus. J Pathol. 2015;235(2):242–52. doi: 10.1002/path.4445 ; PubMed Central PMCID: PMC4268314. - DOI - PMC - PubMed
    1. Henrickson KJ. Parainfluenza viruses. Clin Microbiol Rev. 2003;16(2):242–64. doi: 10.1128/CMR.16.2.242-264.2003 ; PubMed Central PMCID: PMC153148. - DOI - PMC - PubMed
    1. van den Hoogen BG, Herfst S, Sprong L, Cane PA, Forleo-Neto E, de Swart RL, et al.. Antigenic and genetic variability of human metapneumoviruses. Emerg Infect Dis. 2004;10(4):658–66. doi: 10.3201/eid1004.030393 ; PubMed Central PMCID: PMC3323073. - DOI - PMC - PubMed

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