Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines Yuan Lua, John P. Welsha, and James R. Swartza,b,1 Departments of aChemical Engineering and bBioengineering, Stanford University, Stanford, CA 94305 Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved November 15, 2013 (received for review May 7, 2013)

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accination is the most effective way to protect against influenza virus infection, with most of the neutralizing antibodies recognizing the hemagglutinin (HA) protein on the surface of the virus. As of 2007, all commercial influenza vaccines were produced in embryonated chicken eggs, but the generation of a new vaccine takes six to eight months, making it ineffective for combating potential pandemics (1). In 2007, to partially address this concern, the European Union approved Optaflu, a vaccine produced by Novartis using a mammalian cell line (2). In 2013, the recombinant HA vaccine (Flublok) manufactured in insect cells by Protein Sciences was also licensed in the United States (3). However, the new processes would still likely only improve production times by several weeks (4). Hence, rapid production (for example in 1–2 mo) of large amounts of vaccine to stop an epidemic/pandemic remains an important objective. This challenge can be addressed in two ways, either by producing a more broadly protective vaccine to lower the probability of a pandemic occurrance or by developing new, rapid, and scalable technologies for vaccine production. This work seeks to address both. The trimeric HA ectodomains consist of a head domain and a stem domain (Fig. 1A). The assembled HA ectodomain also consists of HA1 and HA2 domains, which are defined by a specific proteolytic cleavage (5). The majority of protective antibodies bind to the HA globular head domain. Consequently, new viral strains arise with the most common mutations occurring in the head domain, thereby avoiding initial antibody suppression. www.pnas.org/cgi/doi/10.1073/pnas.1308701110

Because the HA head domain evolves with a high mutation rate, the influenza vaccine has to be regularly updated. The stem domain, on the other hand, is much more conserved (SI Appendix, Fig. S1) (6, 7). At low pH (5∼6), as the influenza virus is trafficking in endosomal vesicles, the trimeric HA stem domain undergoes a conformational change to trigger fusion to the endosomal membrane, thereby enabling RNA release and successful infection (8). Several research groups have devised constructs consisting of portions of this stem domain in hopes of producing a broadly protective influenza vaccine (1, 9, 10). However, none have produced properly folded stem trimer. Recent work suggests that antibodies generated against this stem domain can cross-react between different virus subtypes, lending hope toward using this domain as an antigen for broadly protective influenza vaccines (10–13). A vaccine, multivalent against several stem subtypes, would not need annual updates and could dramatically reduce the risk of influenza pandemics. The natural production mechanisms suggest that HA stem production would be technically challenging. The first challenge is that the HA stem domain has not evolved to fold and trimerize as an independent unit. Further, its contemporaneous folding along with that of the head domain most likely occurs cotranslationally as first part of the stem, then the head, and finally the rest of the stem domain are extruded from the ER membrane and are orientated by this association. During this process, disulfide bonds must form within each monomer while avoiding intermonomer linkage. Finally, the absence of the head domain exposes internal HA polypeptides that may now be disordered, hydrophobic, or otherwise inappropriate as surface epitopes for Significance The discovery of neutralizing antibodies that block influenza infection by binding to the hemagglutinin (HA) stem domain raised the hope for broadly protective vaccines. These could avoid the need for annual vaccinations and reduce pandemic threats, and the stem subdomain of the trimeric HA ectodomain would be an ideal antigen. However, its production has proven extremely difficult. Here, we describe a simple procedure resulting in high yields of HA stem trimer recognized identically by a panel of neutralizing antibodies as compared with recognition of the full-length HA ectodomain. Cell-free protein synthesis is followed by a simple refolding procedure to produce a rationally mutated stem in which newly exposed protein surfaces are modified and trimerization is induced and covalently stabilized. Author contributions: Y.L., J.P.W., and J.R.S. designed research; Y.L. and J.P.W. performed research; Y.L. contributed new reagents/analytic tools; Y.L., J.P.W., and J.R.S. analyzed data; and Y.L., J.P.W., and J.R.S. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1308701110/-/DCSupplemental.

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The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/ 05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.

Fig. 1. (A) Structural features of the influenza virus HA trimer and its head and stem domains. HA1 regions are shown in blue, and HA2 regions are in cyan. The figure was derived from the X-ray structure of HA from the (H1N1) isolate A/California/05/2009 (H1N1) (PDB ID code 3LZG) and was drawn by using the program PyMOL. Protein yields (B) and autoradiograms (C) of nonreducing SDS/PAGE gels of CFPS products. The arrow indicates the monomer. Stem: no trimerization domain, 32 kDa; Stem-foldon: with the foldon trimerization domain, 36 kDa; Stem-CAT: with the CAT trimerization domain, 58 kDa; S, soluble; T, total protein. Autoradiogram from nonreducing SDS/PAGE gels of refolded proteins (D) and pI analysis of HA protein fragments (E). Polypeptides were refolded with different pH environments (pH 6.0, 8.0, or 10.5) and with (+) or without (−) Tween 20 (0.05%). The theoretical pI values were calculated by using the program ProtParam. All of the residue numbers were in H3 numbering.

stable and soluble protein. Because of these complications, in vivo Escherichia coli expression produced low yields of only 2 mg/L, and the HA stem polypeptides accumulated as inclusion bodies (1). Although some refolding methods have been attempted (14–16), the recovery yields of soluble products have been low, and properly folded stable trimeric assembly has not been confirmed. In this study, we demonstrated the production of HA stem domain from the 2009 pandemic H1N1 strain by using E. colibased cell-free protein synthesis (CFPS) production methods followed by protein refolding. For inducing the trimerization of the HA stem domain, either a CAT or foldon domain was fused to the C terminus. To reduce the chance of the formation of intermolecular disulfide bonds, several HA stem domain mutations were introduced, including removal of hydrophobic and positively charged residues and decreasing the number of disulfide bonds. Protein refolding methods were also optimized. The trimer was further stabilized by introducing disulfide bonds between foldon domains and in the distal region of the stem trimer. The correct immunogenic conformation of this stabilized HA stem trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem trimer at a site that inhibits the structural changes required for membrane fusion. Production consistency and high recovery yields were also demonstrated. Results and Discussion Production and Refolding of HA Stem Domain Trimer. The stem domain of the HA protein is considerably more conserved than 126 | www.pnas.org/cgi/doi/10.1073/pnas.1308701110

the head domain (1, 9). HA is classified into 17 subtypes (H1– H17) based on antigenicity and sequence diversity (17), and, in particular, the stem domains in the same subtype are highly conserved (SI Appendix, Fig. S1). Two subtypes of influenza A, H1N1 and H3N2, most commonly infect humans. Therefore, for example, a vaccine presenting these two stem domains could serve as a broadly protective vaccine. In historical records of previous influenza pandemics, H1N1 influenzas occurred most often and were severe, so the HA stem domain from the influenza virus A/California/05/2009 (H1N1) was chosen as the first target. It is highly likely that the HA stem domains from other subtypes could also be produced by using the same protocol described in this work. Because HA is active as a trimer on the viral surface, two different sequences were fused to the C terminus of the stem domain to induce trimerization. The first was a 29-aa “foldon” sequence that forms a βpropeller structure comprising the C terminus of the fibritin domain of the T4 bacteriophage (18). This domain has been used as a trimerization domain for HA (12, 19). The second candidate trimerization domain was chloramphenicol acetyl transferase (CAT), which expresses and folds well in typical CFPS reactions and also naturally forms a trimer (20). Both domains were linked to the HA sequence by a cleavable tobacco etch virus (TEV) protease site and also contained a His6 tag at the C terminus. The E. coli-based CFPS system was used for this study because it has many advantages for rapidly screening protein constructs compared with traditional E. coli expression systems (21). First, direct use of plasmids or PCR templates as expression templates avoids time-consuming molecular cloning steps. Second, Lu et al.

Mutations of Hydrophobic Regions. Based on these considerations, five sets of mutations were designed (Fig. 2A) to either mitigate newly exposed hydrophobicity (SI Appendix, Fig. S2), reduce the potential for intermolecular ion pairing, or both. Five different groups of mutations were evaluated, M1 (I69T + I72E + I74T + C77T), M2 (I69T + I72E + I74T + C77T + F164D), M3 (I69T + I72E + I74T + C77T + F164D + L174D), M4 (F164D), and M5 (F164D + L174D). The mutations decreased the pIs of two newly exposed stem domain fragments (Stem-HA1-Fragment1 and Stem-HA2-Fragment1) (SI Appendix, Table S1). It should be noted that the targeted mutations are distant from the surface recognized by the neutralizing antibody (12). However, the CFPS soluble yields of these mutants were still low (SI Appendix, Fig. S3). The insoluble inclusion bodies were then refolded by using the dialysis refolding protocol at pH 10.5 (SI Appendix, SI Materials and Methods), and the products were evaluated using size-exclusion HPLC (Fig. 2B). Mutants M3 and M5 produced much fewer aggregates than the wild-type or other variants. The most influential mutations appeared to be F164D + L174D. Therefore, mutant M5 (F164D + L174D) was used for further development. Improving Protein Refolding at pH 8.0. Although the high pH (10.5) folding conditions and the M5 mutation increased the soluble fraction after refolding, when the pH was then lowered to 8.0, the product still became insoluble. We therefore focused on adjusting components in the dialysis buffer to improve folding at pH 8.0, thereby avoiding the need for subsequent pH change. Many factors can affect protein refolding, such as pH, chaotropic agent choice, -S-H/S-S redox ratio, ionic strength, buffer choice, and low molecular weight additives such as arginine and detergents. Detergent Brij 35 was more effective than Tween 20 as judged by SDS/PAGE and size-exclusion HPLC analysis (SI Appendix, Fig. S4). Because the HA stem domain protein contains four disulfide bonds, the choice of sulfhydryl/disulfide redox buffer was next examined. The cystamine/cysteamine system was more effective than GSSG/GSH (SI Appendix, Fig. S5). Cystamine and cysteamine are smaller molecules than GSSG and GSH and possibly can more readily diffuse into the protein structure to assist in disulfide formation and interchange. To identify the best refolding conditions while evaluating seven different factors, an orthogonal (design of experiment) procedure was designed with the seven factors evaluated at two levels (SI Appendix, Table S2). The seven factors are buffer choice (50 mM Tris or 50 mM histidine), [Brij 35] (0% or 0.03%), APPLIED BIOLOGICAL SCIENCES

milligrams of protein can be produced in a few hours in multiwell plates and then directly purified without a cell-lysis step. Third, the lack of a cell wall barrier allows for easy manipulation of reaction conditions, including the incorporation of isotopically labeled amino acids. The E. coli-based CFPS system is linearly scalable from micro-scale (20 μL) to large-scale reactions (100 L) (22, 23) with nearly identical process performance. The system has also been shown to produce active pharmaceutical proteins (22–24). Initial microscale (20 μL) CFPS reactions tested expression of the HA stem construct either alone or with the trimerization domains. These reactions were conducted at 30 °C for 6 h and produced high total yields but much lower soluble yields (Fig. 1B). Nonreducing SDS/PAGE with autoradiography (Fig. 1C) indicated no significant accumulation of soluble monomer, and the higher molecular weight species indicated the formation of incorrect intermolecular disulfide bonds. Refolding of the inclusion bodies was then attempted, using a dialysis refolding method (25–27) under different pH conditions. Eight molar urea was used to solubilize the inclusion bodies and the HA stem protein was purified by using the His6 tag. Oxidized (GSSG) and reduced glutathione (GSH) (molar ratio 1:4) were added to establish a sulfhydryl/disulfide redox environment for the formation and potential isomerization of correct disulfide bonds. L-arginine (0.5 M) and the detergent, Tween 20 (0.05% wt/vol), were also added to assist in refolding. Three different pH values (6.0, 8.0, and 10.5) were tested (Fig. 1D). At pH 6.0, most of the protein was lost, apparently adhering to the dialysis membrane. At pH 8.0, most reaggregated, whereas at pH 10.5, less aggregation occurred. However, the soluble fraction from the pH 10.5 procedure also aggregated when the pH was reduced to 8.0. These observations prompted us to examine regional isoelectric point (pI) values for the polypeptide calculated by using the program ProtParam (28) (Fig. 1E). The theoretical pI values of Stem-HA1-Fragment1 (pI 8.85), Stem-HA1-Fragment2 (pI 9.10), and Stem-HA2-Fragment1 (pI 8.60) were above 8.5, whereas the pI values of other stem domain fragments were less than 5. The divergent pIs suggested the potential for significant interdomain ionic attractions at neutral pH that would largely be avoided at pH 10.5. Furthermore, because the detergent, Tween 20, reduced aggregation, inappropriate hydrophobic interactions were also suggested. Finally, significant aggregation resulted at all three pHs with the CAT trimerization fusion and this folding partner was abandoned.

Fig. 2. (A) HA stem constructs with different mutations in the exposed hydrophobic region. (B) Size-exclusion HPLC profiles of refolded proteins (wild-type, mutants M1, M2, M3, M4, and M5). Dotted red traces indicated 14C radioactivity after 14C leucine incorporation during CFPS. (C) Three-dimensional structures of mutants M5 and M6. (D) Autoradiogram of nonreducing SDS/PAGE gel of refolded HA stem domain mutants M5 and M6.

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Fig. 3. Introduction of disulfide bonds between foldon monomers. (A) Three-dimensional structures of foldon trimer with six possible disulfide bond positions. Autoradiogram of a nonreducing SDS/PAGE gel (B) and size-exclusion HPLC profiles (C) of refolded HA stem construct mutant M6 (O) and constructs with the six different foldon mutations.

[arginine] (0 mM or 500 mM), [NaCl] (0 mM or 150 mM), [sucrose] (0% or 10%), [glycerol] (0% or 10%) and [urea] (0 M or 0.5 M). The data were analyzed by using IBM SPSS Statistics software. The results showed that Brij 35 was the most important factor for improving the refolding yield (SI Appendix, Table S3). Arginine (500 mM) was also marginally beneficial for suppression of protein aggregation in the refolding process and was included in the refolding buffer. Furthermore, two different refolding protocols, dialysis refolding and dilution refolding, were compared. These two protocols produced similar refolding results (SI Appendix, Fig. S6). The dilution refolding protocol is much faster and more convenient and was used in the subsequent experiments. Although the preceding changes improved protein refolding results, SDS/PAGE analysis indicated that intermolecular disulfide bonds were still impeding proper folding. Targeted Deletions for Further Improvement. To avoid the formation of undesired intermolecular S-S bonds, further reduce surface hydrophobicity and pI, and avoid regions with possibly disordered structure, mutant M5 was further modified by deleting two polypeptide regions (H38 to C43 and C49 to N61) containing hydrophobic residues, two positively charged lysines, and three cysteines (Fig. 2C). Cysteine 77 was also mutated to threonine to remove a cysteine that was then unpaired. Again, the deleted regions are far away from the neutralizing antibody binding region. The number of disulfide bonds in each monomer was thereby decreased from 4 to 2. This mutant was named M6. SDS/PAGE results (Fig. 2D) showed that these modifications greatly decreased the formation of undesired intermolecular disulfide bonds. Stabilization of the Stem-Foldon Trimer. The HA stem trimer may be used as an independent vaccine antigen or could possibly be conjugated to virus-like particles (VLPs) or other carriers. In either case, it will be important for the trimeric form to be stable during manufacturing, storage, and administration. Reliable stability is particularly important during the Cu(I) catalyzed click conjugation reaction. Although the foldon domain proved effective in forming the trimers, we reasoned that the limited number of intermolecular hydrogen bonding attractions in the foldon may not sufficiently stabilize this unnatural ectodomain trimer. To stabilize the HA stem trimer, we introduced new cysteine residues into the foldon domain to potentially form intermolecular disulfide bonds between foldon monomers. Beginning with mutant M6, we introduced cysteines at the six following locations to evaluate which pairs could most effectively form intermolecular disulfide bonds (A: G12-D19; B: R10-D19; C: R10-G20; D: P9R20; E: A14-K18; and F: Y15-R17), as shown in Fig. 3A. SDS/ PAGE results (Fig. 3B) showed that mutant E formed the most disulfide-linked trimer. Size-exclusion HPLC results were consistent with this interpretation (Fig. 3C). 128 | www.pnas.org/cgi/doi/10.1073/pnas.1308701110

To further confirm the formation of intermolecular S-S bonds between foldon monomers, we used the TEV protease cleavage site between the HA stem domain and foldon domain to release the foldon trimer after the refolding procedure. Nonreducing SDS/PAGE (SI Appendix, Fig. S7) of the released foldon domains also showed that version E enabled the best formation of disulfide-linked trimers. This mutant was used in subsequent development. Further Polypeptide and Folding Protocol Adjustments. To increase the proportion of trimer and the completeness of intermolecular disulfide bond formation within the foldon trimer, the refolding conditions were further examined. Using cystamine and cysteamine as the “oxido-shuffling” reagent, we compared several ratios, 1:4, 1:10, and 1:20. During such procedures, oxygendriven oxidation can modify the sulfhydryl/disulfide redox potential over time, and a higher fraction of trimer was obtained anaerobically. We therefore conducted refolding procedures in an anaerobic glove box (SI Appendix, Fig. S8), and the cystamine to cysteamine 1:20 ratio was best (SI Appendix, Fig. S9). The final composition of the modified refolding buffer was as follows: 50 mM Tris·HCl, 600 mM arginine, 2 mM EDTA, 0.5 mM cystamine/10 mM cysteamine, and 0.05% Brij35 at pH 8. Although a higher proportion of trimer was obtained after improving refolding conditions with the M6E mutant, we still observed dimer and monomer bands. In the initial design of the HA stem domain antigen, the foldon domain is separated from the HA stem sequence by a cleavable TEV protease site and the fusion protein also has a C-terminal His6 tag. We hypothesized that the TEV protease site and the direct linkage to the His6 tag could have impeded the trimer formation or formation of the intermolecular S-S bonds in the foldon trimer. To structurally isolate the foldon domain, stem trimer mutant M6 with foldon mutation E (M6E) was modified by replacing the TEV protease

Fig. 4. Newly designed HA stem construct (M6EL) based on mutant M6 with foldon mutation E (M6E). (A) Schematic diagram of new HA stem construct design. (B) Autoradiogram of nonreducing SDS/PAGE gel of refolded M6EL HA stem trimer.

Lu et al.

Fig. 5. Introduction of intermolecular disulfide bonds between stem monomers in the stem trimer. (A) The position of newly introduced intermolecular S-S bonds between stem monomers. (B) Protein yields and overall recovery yields of stem construct mutants (M6L and M6EL with the S-S mutations SS1, SS2, SS3, or SS4) after protein expression, purification, and refolding. (C) Autoradiogram of a nonreducing SDS/PAGE gel of refolded HA stem construct mutants. (D) Size-exclusion HPLC profiles of refolded HA stem construct mutant M6EL and M6EL(SS2).

Additional Stabilization of the Stem Trimer. Although the HA stem trimer had been stabilized with disulfide bonds between foldon monomers (Fig. 3) at the membrane proximal end of the molecule, we reasoned that stability could be further improved by also introducing disulfide bonds between stem monomers at the distal end of the protein structure. Cysteines were introduced in the Stem-HA2-Fragment3 (Fig. 1E) at the following locations (SS1: I192C-W193C; SS2: G188C-F189C; SS3: K184C-V185C; SS4: L181C-N182C), as shown in Fig. 5A. Cysteine adjacency was chosen to discourage intramolecular bonds in favor of bonds between monomers. Sites were also chosen based on natural intermolecular proximity. Disulfide bond formation at these sites could both encourage and help confirm proper trimer formation. SDS/PAGE results (Fig. 5C) showed that all four designs formed intermolecular disulfide bonds between stem monomers in the absence of foldon mutation E. The SS2 mutant of M6EL showed the highest protein yield (363 μg/mL CFPS reaction volume; 27% higher than M6EL) after protein expression, purification, and refolding (Fig. 5B). The mutant M6EL(SS2) was used in subsequent studies. Size-exclusion HPLC results (Fig. 5D) further confirmed better trimer formation for M6EL(SS2). Confirming Neutralizing Antigen in Stabilized HA Stem Trimer. The correct immunogenic conformation of this stabilized HA stem trimer M6EL(SS2) was confirmed by using antibodies CR8020, CR6261, C179, and FI6 known to be specific for neutralizing epitopes located in the HA stem domain. An ELISA was used with green fluorescence protein (GFP) as the negative control. The full-length H1 HA monomer [A/California/07/2009(H1N1)] and the full-length H1 HA trimer [A/South Carolina/1/1918 (H1N1)] (with foldon trimerization domain) were used as positive controls. ELISA binding results (Fig. 6) showed that the new H1 HA stem trimer and the full-length H1 HA trimer control were recognized with nearly identical affinity by all four antibodies. As expected, recognition of the full-length HA monomer differed significantly from that of the trimers. The FI6 antibody bound HA monomer more strongly than the trimer, whereas CR6261 and C179 bound the trimer more strongly. CR6261, C179, and FI6 all bind HAs belonging to the H1 subtype, but CR8020 only can bind HAs belonging to H3 and H7 subtypes (29). Together these results demonstrate that the M6EL(SS2) HA stem antigen is properly folded. Lu et al.

Conclusion In this work, we describe an E. coli-based CFPS method for producing an HA stem domain trimer assembly derived from the 2009 pandemic H1N1 strain. The CFPS production of this antigen potentially provides a rapid, scalable, and cost-effective means to combat pandemic and epidemic threats when a rapid response and large quantities of vaccine are required. Although the HA stem antigen by itself may not be an effective vaccine, it can now be tested with adjuvants or by attaching it to virus-like particles to enhance its immunogenicity and increase protective efficacy. We also developed an effective refolding method to produce the correctly folded trimeric HA stem domain protein. We suggest this stepwise procedure may provide a valuable paradigm for producing properly folded multimeric domains of membrane-associated cell and viral surface proteins. To reduce the aggregation and the formation of incorrect intermolecular disulfide bonds, several newly exposed hydrophobic residues were replaced, the isoelectric points of newly exposed protein fragments were decreased, and the number of cysteines in the HA stem domain was reduced from eight to four. The HA stem trimer was further stabilized by introducing intermolecular disulfide bonds between foldon monomers and between stem domain monomers. The refolded and stabilized trimeric HA stem domain protein was recognized identically compared with full-length HA ectodomain by neutralizing antibodies CR6261, C179, and FI6, suggesting that the neutralizable epitope is

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target sequence with a (GS)3 linker, and another (GS)3G linker was added between the foldon domain and the His6 tag, as shown in Fig. 4A. This HA stem design was named “M6EL.” Nonreducing SDS/PAGE results (Fig. 4B) showed that the addition of the (GS)n linkers increased the proportion of trimer to approximately 80%. Repeated production batches indicated excellent reproducibility (SI Appendix, Table S4).

Fig. 6. ELISA of HA stem construct M6EL(SS2) using antibody CR8020 (A), CR6261 (B), C179 (C), and FI6 (D). Green fluorescence protein (GFP) protein with a C-terminal His6 tag was used as the negative control. The full-length H1 HA monomer [A/California/07/2009(H1N1)] (with His6 tag) and the fulllength H1 HA trimer [A/South Carolina/1/1918(H1N1)] (with foldon trimerization domain and His6 tag) were used as the positive controls.

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properly folded. This influenza HA stem trimer therefore is an excellent candidate for the design and production of broadly protective influenza vaccines. Based on the insights gained, we believe that similar procedures can be rapidly developed for the other influenza HA stem domains needed for universal vaccines. As mentioned, this overall protocol may provide a paradigm for the rational design and evaluation of stable properly folded domains of other multimeric proteins. The protocol begins with cell-free protein expression and then involves four main steps. Of course, the development of any successful folding protocol benefits when the starting material is relatively pure, and a rapid and precise assessment method for protein folding is essential. i) Analyze the pI of protein fragments particularly on newly exposed protein surfaces. Significant pI differences between fragments could result in significant intermolecular ionic attractions and protein aggregation at neutral pH, which could inhibit folding. Mutations to reduce or alter charges on new surfaces may be beneficial. ii) Analyze the hydrophobicity at the newly exposed protein surfaces. Newly exposed hydrophobic regions could result in undesired protein aggregation in the protein folding process. Mutating these exposed hydrophobic residues to hydrophilic residues could simultaneously reduce surface hydrophobicity and the pI differences of polypeptide domains. If possible, targeted mutations should be distant from the biologically relevant regions of protein. iii) Introduce intermolecular covalent disulfide bonds between monomers to stabilize the multimeric protein. The disulfide bonds can form where the distance between the two residues is expected to be less than 8.5 Å. Again, if possible, these modifications should be introduced at points distant from the region of the protein with important functional properties. iv) Optimize the refolding conditions, including pH, disulfide exchange reagents, buffer additives (arginine, salts), divalent 1. Bommakanti G, et al. (2010) Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proc Natl Acad Sci USA 107(31):13701–13706. 2. Extance A (2011) Cell-based flu vaccines ready for US prime time. Nat Rev Drug Discov 10(4):246–247. 3. Yang LPH (2013) Recombinant trivalent influenza vaccine (flublok(Ò)): A review of its use in the prevention of seasonal influenza in adults. Drugs 73(12):1357–1366. 4. Wright PF (2008) Vaccine preparedness—are we ready for the next influenza pandemic? N Engl J Med 358(24):2540–2543. 5. Zhirnov OP, Ikizler MR, Wright PF (2002) Cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases. J Virol 76(17):8682–8689. 6. Chen GL, Subbarao K (2009) Attacking the flu: Neutralizing antibodies may lead to ‘universal’ vaccine. Nat Med 15(11):1251–1252. 7. Ikonen N, et al. (2010) High frequency of cross-reacting antibodies against 2009 pandemic influenza A(H1N1) virus among the elderly in Finland. Euro Surveill 15(5): 16–23. 8. Gaudin Y, Ruigrok RWH, Brunner J (1995) Low-pH induced conformational changes in viral fusion proteins: Implications for the fusion mechanism. J Gen Virol 76(Pt 7): 1541–1556. 9. Steel J, et al. (2010) Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio 1(1):e00018–e10. 10. Wang TT, et al. (2010) Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes. Proc Natl Acad Sci USA 107(44):18979–18984. 11. Li GM, et al. (2012) Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells. Proc Natl Acad Sci USA 109(23):9047–9052. 12. Ekiert DC, et al. (2009) Antibody recognition of a highly conserved influenza virus epitope. Science 324(5924):246–251. 13. Wei CJ, et al. (2010) Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329(5995):1060–1064. 14. Swalley SE, et al. (2004) Full-length influenza hemagglutinin HA(2) refolds into the trimeric low-pH-induced conformation. Biochemistry-Us 43(19):5902–5911. 15. Curtis-Fisk J, Spencer RM, Weliky DP (2008) Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein. Protein Expr Purif 61(2):212–219.

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cation (or EDTA), and mild detergents (Tween 20, Brij 35), using a design of experiment approach. Materials and Methods Design and Construction of HA Stem Domain Construct. The HA stem domain protein was from the influenza virus A/California/05/2009 (H1N1) hemagglutinin (accession no. ACP41926). The sequences of all HA stem constructs are shown in SI Appendix, SI Materials and Methods. CFPS. CFPS was conducted by using the PANOx-SP (phosphoenolpyruvate, amino acids, nicotinamide adenine dinucleotide, oxalic acid, spermidine, and putrescine) cell-free system as described (30, 31) (SI Appendix). Protein Purification and Refolding. After CFPS reactions, the denatured proteins were purified by a Ni-NTA column (Qiagen). Two different refolding methods were used and compared: dialysis refolding and dilution refolding (SI Appendix, SI Materials and Methods). Size Exclusion HPLC. HA stem domain proteins (mutants M1, M2, M3, M4, and M5) refolded at pH 10.5 were initially analyzed by using a Discovery Bio GFC 500 HPLC column (Sigma-Aldrich). Unless specifically stated otherwise, other refolded HA stem domain proteins were tested by using an Ultrahydrogel 500 HPLC column (Waters). See SI Appendix for details. ELISA Binding of HA Stem Constructs. Antibodies CR8020, CR6261, C179, or FI6 were coated on 96-well ELISA plates (NUNC MaxiSorp) for ELISA tests. Fulllength HA monomer [A/California/07/2009(H1N1) and full-length HA trimer (A/South Carolina/1/1918(H1N1)] were used as the positive controls. GFP protein was used as the negative control. See SI Appendix for details. ACKNOWLEDGMENTS. We thank Prof. Mark M. Davis for help in providing the initial funding during the 2009 influenza pandemic and Dr. Jeffrey Boyington and the National Institutes of Health (NIH) Vaccine Research Center for providing antibodies and HA proteins. This work was supported through the NIH-funded Stanford U19 consortium Grants AI057229 and AI090019.

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Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines.

The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect agai...
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