Design and initial characterization of bRSV F immunogens

The RSV F glycoprotein is conserved between bRSV and hRSV, with sequence identities of ~80% (ref. 2) (Fig. 1b, d, Supplementary Fig. 1 and Supplementary Table 1), and multiple F-directed antibodies can neutralize both hRSV and bRSV.21,22,23,24 Based on the success of our prior engineering of pre-F hRSV F trimer,17 we modified bRSV F to create thermostable pre-F trimers. A disulfide between residues 155 and 290 (DS) along with cavity-filling mutations S190F and V207L (Cav1) and a C-terminal T4-phage fibritin trimerization domain (foldon) were incorporated into bRSV F from seven different strains to make bovine versions of DS-Cav1 (ref. 17) (bDS-Cav1s) (Fig. 1a, b and Supplementary Table 2), which included cleavable C-terminal His and Strep tags for purification. Upon expression in Expi293F cells only three of the seven bDS-Cav1s (strains 391-2, ATue51908, and RB94, respectively) expressed at greater than 0.5 mg/l of culture (Supplementary Table 2). All three of these bDS-Cav1s were recognized by pre-F-specific mAbs D25 (ref. 20) and MPE8 (ref. 21) as well as by mAb motavizumab (Mz)25 (Supplementary Table 2).

To enhance immunogenicity, we next sought to optimize bDS-Cav1 thermostability. To minimize the number of designs evaluated, we selected two RSV strains (391-2 and RB94) to optimize initially, with the intent to introduce the best mutations from the final set into the third strain, ATue51908. Previous investigations18, 26,27,28, – 29 of type I fusion machines have indicated that removal of the cleavage site to create sc variants can improve pre-F stability. Furthermore, the introduction of interprotomer disulfide bonds (DS2) has been observed to enhance both stability and immunogenicity of hRSV F immunogens.18 Therefore, with a focus on these two aforementioned types of stabilizations, 90 variants of bDS-Cav1 were designed, all of which employed an sc topology and 32 of which contained an interprotomer disulfide (DS2 variants). Additionally, many of the 90 designs incorporated internal cavity-filling mutations, core residues from hRSV F for increased stability, and additional sites of N-linked glycosylation to mask irrelevant epitopes.

All 90 bDS-Cav1 designs were evaluated for expression and antigenic recognition by mAbs D25, MPE8 and Mz in a 96 well-microplate transient transfection format.17 Each design was scored by summing enzyme-linked immunosorbent assay (ELISA) readings for the pre-F-specific mAbs D25 and MPE8 (Supplementary Tables 3 and 4). The top three-scoring DS2 designs (DS2-v1, DS2-v5, and DS2-v3) and the top two-scoring 391-2 and RB94 sc designs without interprotomer disulfides (sc-v1 and sc-v4) were selected for additional evaluation (Supplementary Tables 3 and 4). To expand the dimensions of our search for optimal immunogens, we mixed and matched DS2 interprotomer disulfides (Q98C Q361C, A149C Y458C, and N183GC N428C) and sc formats (sc9 and sc9-10) from the top five-scoring designs and added the ATue51908 strain to generate additional designs for a total of nine constructs, which were expressed in 1 liter Expi293F cultures (Supplementary Table 5). The sc designs sc9 and sc9-10 differed only in two residues, with a GS linker replacing F 2 F 1 sites of cleavage and fusion residues 106–144 or 104–144, respectively.18 Of these nine designs, the two sc-only variants gave 7–9-fold higher expression yields compared to the other variants (Supplementary Table 5). However, size exclusion chromatography (SEC) indicated their respective molecular sizes were larger than expected, suggesting unfolding or aggregation. We, therefore, chose three of the remaining five DS2 designs (each with sc topology and added interprotomer disulfides) with the highest yield (Supplementary Table 5) for immunogenic, antigenic, physical, and structural characterizations. Additionally, as benchmarks, we used the DS-Cav1 variant of each of the three strains and also the post-F form of each of the three strains (the latter created by removing the RSV F fusion loop residues 137–146), as previously described.30 Altogether, the sc-DS2, DS-Cav1 and post-F immunogens of each of the three strains totaled nine final immunogen constructs.

All nine of these constructs gave expression yields of 2–5 mg/l except for DS2-v35 (0.24 mg/l) and RB94 DS-Cav1 (0.76 mg/l) (Table 1), and the post-F forms consistently gave the highest expression (3–5 mg/l). After purification on nickel and Strep-Tactin affinity columns, and subsequent cleavage of C-terminal affinity tags, all nine immunogens behaved well when analyzed by SEC. Post-F forms eluted at slightly larger sizes than pre-F forms due to their elongated shape,20, 30 and pre-F forms all eluted with peaks consistent with trimer formation (Supplementary Fig. 2a, b).

Table 1 Antigenic and physical characterization of bRSV F glycoprotein immunogens Full size table

Antigenic characteristics of bRSV F immunogens

The antigenicity of each purified immunogen was evaluated with biolayer interferometry to assess recognition by the antigenic site Ø-directed mAb D25 (ref. 20), antigenic site II-directed mAb Mz25, 31 and quaternary-specific mAbs AM14 (ref. 19) and MPE8 (ref. 21) (Table 1). The three pre-F-specific mAbs, D25, MPE8, and AM14, recognized all six immunogens containing pre-F stabilizing mutations, confirming stabilization of their pre-F conformations. Moreover, recognition by the quaternary-specific mAbs MPE8 and AM14 substantiated the formation of native-like trimers. In contrast, these mAbs did not recognize any of the post-F immunogens. As expected, Mz recognized all nine immunogens since its site II epitope is not affected by pre- and post-F conformational changes. D25 recognized the pre-F immunogens with affinities ranging from 0.4–420 nM, suggesting that antigenic site Ø may be adversely affected by some of the stabilizing mutations. The three DS-Cav1-only immunogens which also had the least number of mutations had the highest affinity for D25. Notably, all mAbs except for D25 recognized the pre-F immunogens with nanomolar affinity (0.2–12.8 nM) even though these mAbs were elicited by hRSV.

Physical characteristics of bRSV F immunogens

We next assessed the stability of purified immunogens by subjecting them to extremes of temperature, pH, and osmolarity as well as cycles of freeze-thaw and quantifying their subsequent recognition by D25 (for pre-F) or Mz (for post-F) (Table 1). All nine immunogens were observed to generally tolerate pH and osmolarity extremes and freeze-thaw cycles, consistent with data reported for hRSV DS-Cav1 (ref. 17). The three DS-Cav1-only immunogens, 391-2 DS-Cav1, ATue51908 DS-Cav1 and RB94 DS-Cav1, were most susceptible to physical extremes and lost 25–60% of their D25 reactivity upon exposure to high (3M) osmolarity. Curiously, the majority of immunogens actually increased reactivity to D25 or Mz after exposure to high pH. Not surprisingly, all three post-F immunogens, were stable at higher temperatures as measured by Mz affinity. Although none of the DS-Cav1-only immunogens were able to antigenically survive exposure to 70 °C, consistent with that observed for hRSV F pre-F-stabilized immunogens,17, 18 all three of the DS2 immunogens tolerated exposure to high temperature.

Structural characteristics of bRSV F immunogens

To further confirm the pre- and post-F conformations of the engineered bRSV F immunogens, we examined them by negative stain electron microscopy (EM), followed by reference-free two-dimensional (2D) class averaging of the images (Supplementary Fig. 2c). As expected, each of the pre-F immunogens displayed bulb-like trimer structures with a short stem-like structure at one end, whereas the post-F immunogens displayed longer and more slender structures, each of which was consistent with known crystal structures of pre- and post-F hRSV F.17, 20, 30, 32

The crystal structures of two pre-F-stabilized bRSV F immunogens, ATue51908 DS-Cav1 and DS2-v1 were determined to 2.65 and 3.50 Å resolution, respectively (Supplementary table 6). ATue51908 DS-Cav1 crystallized in a monoclinic lattice not previously observed with hRSV F immunogens. The overall structure of ATue51908 DS-Cav1 was similar to that of hRSV DS-Cav1 (PDB ID 4MMU)17 with a root mean square deviation (rmsd) of 1.0 Å for 437 Cα atoms excluding residues 209–215 in a membrane distal loop adjacent to antigenic site Ø (Fig. 2a). In this crystal form, the appended C-terminal foldon trimerization domain was visible in the electron density map, although its threefold axis was tilted by ~17 degrees relative to the threefold axis of bRSV pre-F due to crystal packing (Fig. 2a). The introduced DS and S190F mutations showed strong electron density, while V207L showed weaker but detectable electron density (Fig. 2a). The DS2 immunogen DS2-v1 crystallized in the cubic lattice commonly observed with hRSV F pre-F immunogens and, like ATue51908 DS-Cav1, its structure was similar to hRSV DS-Cav1 with an rmsd of 1.1 Å for 435 Cα atoms. Although side chains for the DS-Cav1 mutations were not clearly apparent in the electron density, partly due to the 3.50 Å resolution of the structure, the DS2 interprotomer 98C-361C disulfide and nearby sc linker showed traceable electron density (Fig. 2b). We observed the 98C-361C disulfide bond to cause a local distortion of the α1 helix (in which 98C is located), although the local structure surrounding 361C was unperturbed. Comparison of the two bRSV pre-F-stabilized structures at the backbone level revealed high-structural similarity with an rmsd of 0.9 Å between 434 equivalent Cα atoms excluding the F 2 F 1 linker region. The greatest structural differences between the hRSV and bRSV pre-F immunogens were observed in residues 206–215 at the apical loop between α4 and α5 near antigenic site Ø (Fig.2a, b, left panels), which is also the region of highest sequence divergence (only ~50% identity) between the two species (Supplementary Fig. 1). These structural and sequence difference may explain the lower-binding affinity of D25 to bRSV pre-F (Table 1) relative to hRSV pre-F.17 Overall, the structural analysis confirmed the similarity of bRSV and hRSV pre-F structures, with subtle differences in specific regions, including a loop near antigenic site Ø.

Fig. 2 Crystal structures of pre-F-stabilized bRSV F immunogens. a Crystal structure of bRSV F ATue51908 DS-Cav1 depicted by a Cα-worm representation color-coded by atomic mobility factors, with thick, red worm for flexible regions and thin, blue worm for more rigid regions. Atomic level details are shown in insets on the right with stick representations and 2Fo-Fc electron density (blue) for regions that were mutated to stabilize the pre-F conformation. The upper left inset shows a ribbon superposition of the antigenic site Ø region of ATue51908 DS-Cav1 (lime) with the structure of hRSV F DS-Cav1 (gray; PDB ID 4MMU).17 b Crystal structure of the DS2 immunogen bRSV F DS2-v1, depicted as in a Full size image

Immunogenic characterization of bRSV F immunogens in mice

To evaluate immunogenicity, each of the nine bRSV F immunogens was used to immunize a group of 10 CB6F1/J mice. Each immunogen dose comprised 10 μg of protein adjuvanted with 50 μg of polyinosinic:polycytidylic acid (Poly I:C). Mice were primed and boosted intramuscularly at weeks 0 and 3, respectively. Analysis of week 5 sera revealed geometric mean reciprocal EC 50 neutralization titers of 6880–11,453 for pre-F immunogen-immunized mice, which were 33- to 55-fold higher (P < 0.0001) than the titers (geometric mean 100–210) observed for the post-F immunogen-immunized mice (Fig. 3a and Supplementary Table 7). Neutralization titers elicited from pre-F-immunized mice were 82–110-fold greater than the calibrated protective titer of 100 (ref. 17). Although DS2-v1 elicited the highest titers (geometric mean 11,453), all of the pre-F immunogen-elicited titers were statistically comparable with each other. Thus the 1000-fold difference in D25 mAb-binding affinities observed between various pre-F immunogens (Table 1) did not appear to impact the neutralization titers of elicited sera. To gauge the overall immunogenicity of each immunogen, the sera binding response to pre-F and post-F immunogens was assessed by ELISA (Supplementary Fig. 3a). Similar to the neutralization results, the binding titers of pre-F elicited sera to the six pre-F immunogens were statistically comparable to each other with geometric mean end point-binding titers ranging from 4687 to 100,323. Intriguingly, sera from DS2-v1-immunized mice displayed the lowest titers for pre-F immunogen even though it had the highest titers of neutralizing antibodies. As expected, sera elicited by pre-F immunogens displayed lower-binding titers to post-F immunogens.

Fig. 3 Serum neutralizing antibody titers elicited by engineered bRSV F pre-F trimers. Pre-F-stabilized bRSV F glycoproteins elicited geometric mean EC 50 neutralization titers between 43–344-fold higher than post-F in mice and calves, respectively. Schematic immunization procedures for bRSV F variants in seronegative mice (a) and calves (b). Neutralization titer from each animal is shown as an individual dot, and geometric means are indicated by black horizontal lines. Immunization groups are color-coded. Lod, limit of detection (titer = 100) is indicated with a horizontal dashed line. Vertical dotted lines separate immunogen strains in a and weeks post prime in b. Serum antibody binding ELISA data is summarized in Supplementary Fig. 3. P values were determined by two-tailed Mann–Whitney tests. *Indicates P ≤ 0.05, **indicates P ≤ 0.01, ***indicates P ≤ 0.001 and ****indicates P ≤ 0.0001. There are 10 mice per group for the mouse immunizations. For calf immunizations, the DS2-v1 and post-F groups each contained five animals and the placebo group contained four animals Full size image

Immunogenic characterization of bRSV F immunogens in calves

To investigate the effectiveness of pre-F-stabilized RSV F vaccines in bRSV-seronegative calves, we selected the highly stable DS2 immunogen (DS2-v1), which in mice elicited the highest neutralization titers (geometric mean reciprocal EC 50 11,453). As controls we chose post-F 391-2 and used phosphate-buffered saline (PBS) to immunize a placebo group. Groups of five 3–6-week-old male calves (Supplementary Table 8) were immunized twice at weeks 0 and 4, and sera were collected 2 weeks after each immunization. Each injection consisted of 50 μg protein in 0.6 ml PBS adjuvanted with 1.4 ml of an oil-in-water adjuvant ISA 71G. The reciprocal EC 50 neutralization titers from the DS2-immunized calves were observed to increase exponentially at weeks 2, 4, and 6 relative to week 0, resulting in a final geometric mean titer of 56,055 2 weeks after the boost (Fig. 3b and Supplementary Table 9). At week 8, 4 weeks after the boost, titers dropped to a geometric mean of 21,849. In contrast, by week 8, post-F immunogen elicited minimal titers (geometric mean 172) within the same range as two of the saline control-immunized calves (141 and 505, respectively), which may have had maternally-derived serum antibodies at the start of the study. Results from ELISA analysis of sera from DS2-immunized calves mirrored the neutralization titers with binding titers steadily increasing and then slightly decreasing from weeks 6 to 8 (Supplementary Fig. 3b). As expected, sera from post-F-immunized calves followed a similar trend, reaching geometric mean titers approximately 3.0 and 3.8-fold lower than DS2 by weeks 6 and 8, respectively. It is also clear from the neutralization data (Fig. 3b) that post-F elicited significantly lower levels of neutralizing antibodies. Not surprisingly, sera elicited by DS2 immunogen displayed lower-binding titers to post-F immunogen, and post-F-elicited sera recognized both pre- and post-F with comparable titers (as observed with hRSV DS-Cav1 immunization in NHPs).17 Results from a competition ELISA showed that the week 6 sera from DS2-immunized calves competed with the broadly neutralizing mAbs AM14, D25, RSD5 (ref. 21), MPE8 and palivizumab (Pz),25 suggesting that antigenic sites Ø, II, III, and V were all targeted33 (Supplementary Fig. 4). Although the mAb Mz showed considerably less competition than the other antigenic site II mAb, Pz, its high 34.6 pM affinity25 for RSV F likely limited sera competition. As expected, sera from post-F-immunized calves competed to a much lower extent with the pre-F-specific mAbs, AM14, RSD5, and MPE8, and did not compete with D25. Interestingly, two mAbs compatible with post-F RSV F, Mz and Pz, also competed to a much lower extent with post-F-elicited sera. This suggests that a minority of the post-F-elicited sera was directed against site II. Overall, the results in calves demonstrate the striking superiority of the pre-F stabilized DS2-immunogen vs. post-fusion F, with pre-F-induced neutralizing titers more than 100-fold higher than post-F-induced neutralizing titers by week 6.

bRSV challenge of immunized calves

Next, all calves were challenged by intranasal and intratracheal routes with the heterologous Snook strain of bRSV, 4 weeks after the boost. Calves were monitored daily for clinical signs of disease and for viral titers in the nasopharynx for 6 days after challenge. At day 6 after challenge, calves were euthanized and bronchoalveolar lavage (BAL) and lung biopsies from three regions of the lung were obtained to determine viral titers, neutrophil infiltration, and the extent of microscopic and macroscopic lesions. Remarkably, calves vaccinated with DS2 had no detectable bRSV viral titers in nasopharyngeal secretions (Fig. 4a and Supplementary Table 10). No detectable bRSV titers were observed from a postmortem lung wash, samples of tracheal epithelium, right apical, right cardiac or left cardiac regions of the lung (Fig. 3d and Supplementary Table 11). In contrast, peak nasopharyngeal virus titers 6 days post challenge ranged from 1.81 to 2.70 log 10 pfu/ml in post-F and PBS vaccinated calves (Supplementary Table 10). Likewise, virus was isolated post mortem from BAL cells of all of the post-F-immunized and PBS-immunized calves with the greatest extent of lung virus replication in the PBS-immunized control animals (Fig. 4d). Thus, all DS2-immunized calves were protected from bRSV viral replication in both the upper and lower respiratory tracts. Furthermore, four out of five of the DS2-immunized calves were also protected from clinical signs of disease, lung inflammation and macroscopic lung lesions (Fig. 4b, c, Supplementary Fig. 5 and Supplementary Tables 12–14). Clinical scores, based mainly on differences in respiratory rate (RR) and body temperature, were minimal for most of the pre-F- and post-F-immunized calves. Scores trended higher for the PBS-immunized controls, but were not significantly different from the other two groups (Supplementary Tables 12–13). However, both RR and body temperature increased in all PBS-immunized calves, 6 days after bRSV challenge, whereas the RR increased in only two post-F-immunized and one pre-F-immunized calves at this time (Supplementary Fig. 5a). Although the one calf in the pre-F-immunized group that had developed a raised RR and body temperature also exhibited signs of lung inflammation, the geometric mean number of cells observed in the BAL, the percentage of polymorphonuclear neutrophils (PMNs) in BAL, and the percentage of macroscopic lung lesions were all statistically lower than in the placebo group (Fig. 4b, c and Supplementary Tables 12–14). The post-F-immunized calves displayed intermediate levels of lung inflammation. Although the extent of macroscopic lung lesions in the post-F-immunized calves was less than that observed in the placebo group, the percentage of PMNs in BAL was similar to that seen in BAL from calves in the placebo group. Microscopic lung lesions in the placebo group, 6 days post challenge, were typical of bRSV bronchiolitis and alveolitis and were characterized by epithelial necrosis and hypertrophy of bronchiolar epithelium bronchiolitis, bronchiolar exudate containing desquamated epithelial cells, neutrophils and macrophages, and thickening of alveolar walls due to infiltration by mononuclear cells and granulocytes (Supplementary Fig. 6c). In addition to bronchiolitis and alveolitis, peribronchiolar lymphoreticular hyperplasia were seen in three of the post-F-immunized calves (Supplementary Fig. 6b). In contrast, bronchiolitis and alveolitis were absent from all but one of the DS2-immunized calves, and the histopathology was essentially restricted to a peribronchiolar lymphoreticular hyperplasia (Supplementary Fig. 6a).