Significance The discovery of broadly neutralizing Abs (bnAbs) against infectious agents such as influenza virus and HIV-1 has sparked interest in creating vaccines that focus an Ab response toward a particular epitope of a protein. These “immunofocusing” strategies have shown promise but are also burdened with inherent limitations. We introduce an immunofocusing method called protect, modify, deprotect (PMD) that uses a bnAb as a molecular stencil to create vaccine candidates that direct the immune response toward the epitope of the bnAb. PMD has the potential to provide epitope-specific immunofocusing, in a generalizable manner.

Abstract In creating vaccines against infectious agents, there is often a desire to direct an immune response toward a particular conformational epitope on an antigen. We present a method, called protect, modify, deprotect (PMD), to generate immunogenic proteins aimed to direct a vaccine-induced antibody (Ab) response toward an epitope defined by a specific monoclonal Ab (mAb). The mAb is used to protect the target epitope on the protein. Then the remaining exposed surfaces of the protein are modified to render them nonimmunogenic. Finally, the epitope is deprotected by removal of the mAb. The resultant protein is modified at surfaces other than the target epitope. We validate PMD using a well-characterized antigen, hen egg white lysozyme, then demonstrate the utility of PMD using influenza virus hemagglutinin (HA). We use an mAb to protect a highly conserved epitope on the stem domain of HA. Exposed surface amines are then modified with short polyethylene glycol chains. The resultant antigen shows markedly reduced binding to mAbs that target the head region of HA, while maintaining binding to mAbs at the epitope of interest. This antigenic preference is also observed with yeast cells displaying Ab fragments. Antisera from guinea pigs immunized with the PMD-modified HA show increased cross-reactivity with HAs from other influenza strains, compared with antisera obtained with unmodified HA trimers. PMD has the potential to direct an Ab response at high resolution and could be used in combination with other such strategies. There are many attractive targets for the application of PMD.

Vaccines are among the most profound accomplishments of biomedical science and provide cost-effective protection against infectious disease. Many vaccines work by eliciting a neutralizing Ab response that prevents infection (1). However, for some infectious agents, it has not been possible to create an efficacious vaccine, and for others, the protection provided by vaccines is strain-specific.

In the case of influenza, the majority of Abs elicited by vaccination target the trimeric viral surface glycoprotein, hemagglutinin (HA) (2, 3). The 3D structure of HA consists of two regions, the head and the stem (4). Most of the HA-directed Ab response focuses on the head region, which is therefore considered immunodominant (2, 3). Amino acid residues on the surface of this immunodominant head region vary substantially among different strains and change continuously in a phenomenon referred to as antigenic drift (5). This variability, which leads to new circulating virus strains, coupled with the immunodominance of the head region, necessitates the production of new seasonal vaccines against influenza (5).

Strikingly, there is an epitope within the stem region of HA that is highly conserved among influenza strains and not subject to seasonal variation (6⇓⇓⇓⇓⇓–12), likely because residues that form this epitope are critical for viral fusion mediated by HA (13, 14). Except in rare cases, there is no significant immune response toward the stem region (15). Nonetheless, Okuno et al. (16) isolated an mAb that targets this conserved epitope and demonstrated its broad neutralizing activity. Since the discovery of this broadly neutralizing Ab (bnAb) 26 y ago (16), many other HA stem-binding bnAbs have been characterized (6⇓⇓⇓⇓⇓–12). In addition, expression of such bnAbs protects mice from lethal challenges with a broad range of influenza subtypes (17). Taken together, these results suggest that if Abs targeting the conserved stem epitope could be elicited, it might be possible to create a universal flu vaccine (5, 18⇓–20). Such a vaccine might provide cross-strain protection against all circulating and future pandemic strains (i.e., new strains transmitted from animals to humans, such as those that led to the 1918, 1957, 1968, and 2009 pandemics) of influenza (21).

Toward this goal, there has been substantial interest in directing a vaccine-induced Ab response toward the conserved stem region of HA. This would require avoiding the normal, immunodominant Ab response against the head. Strategies that aim to direct the immune system toward a particular region of a protein are referred to as “immunofocusing” (22).

Previous immunofocusing work, either against influenza or other infectious agents, has used a variety of approaches. The five most prominent examples are (i) epitope masking (23⇓⇓⇓⇓–28), (ii) epitope scaffolding (29⇓⇓⇓–33), (iii) protein dissection (34⇓⇓–37), (iv) antigen resurfacing (38⇓–40), and (v) cross-strain boosting (41⇓–43). Epitope masking is a method that shields the immunodominant region of a protein, often using unnatural glycosylation sites, to discourage Ab formation. Epitope scaffolding aims to transplant a conformational epitope of interest onto a unique protein scaffold. Protein dissection removes undesirable or immunodominant epitopes from the native antigen. Antigen resurfacing uses site-directed mutagenesis to install less-immunogenic residues at regions outside the epitope of interest. Finally, cross-strain boosting uses sequential immunizations with other strains or chimeric proteins that vary at off-target epitopes.

Significant progress has been made with these immunofocusing strategies. These methods have inherent limitations, however. They are not easily generalizable, making application to new antigens challenging. With the exception of epitope scaffolding (which requires extensive protein engineering), these immunofocusing methods are also generally “low-resolution” (i.e., directed toward a region of the protein that is significantly larger than a typical Ab epitope). In addition, with some of these methods, maintaining the precise 3D structure of the epitope can be challenging.

Here we introduce a method, which we call “protect, modify, deprotect” (PMD), that has the potential to provide high-resolution immunofocusing in a generalizable manner with minimal protein engineering. The method uses a bnAb as a molecular stencil to generate an antigen that focuses the immune response toward the bnAb epitope. Although bnAbs have been used previously to inform and guide immunogen design, we are not aware of their use as reagents in the creation of vaccine candidates.

The steps in PMD are (i) protection of an epitope on an antigen by binding of a bnAb, (ii) chemical modification of exposed sites to render them nonimmunogenic, and (iii) deprotection of the epitope of interest by dissociation of the Ab-antigen complex. This produces an immunogen in which the only unmodified region is the epitope mapped by the bnAb (Fig. 1).

Fig. 1. A general schematic of the PMD strategy. First, the epitope is protected by combining the mAb (hashed) with the antigen (white). Then the surfaces of the protein complex are modified to render them nonimmunogenic (shown as darker shading). Finally, the epitope is deprotected by removal of the mAb.

To establish the PMD method, we use hen egg white lysozyme (HEWL), a stable monomeric protein with well-characterized epitopes (44). We protect an epitope on HEWL by binding it to an mAb-conjugated resin (45). We then modify surface amines to add short polyethylene glycol (PEG) chains, which are known to decrease immunogenicity locally (28, 46⇓–48). The modified HEWL derivatives, dissociated from the mAb resin, have antigenic properties consistent with the expected properties based on the location of surface amines in Ab cocrystal structures.

We then use PMD to generate an influenza HA antigen designed to skew the immune response toward a conserved epitope on the stem. We confirm that the PMD-generated HA is properly folded and displays markedly reduced binding to mAbs that target the HA head, while maintaining binding to mAbs that target the stem. We also use the PMD-generated HA as bait in fluorescence-activated cell sorting (FACS) experiments with a polyclonal yeast mini-library displaying single-chain variable fragments (scFvs) and obtain significant enrichment for stem-directed clones. Finally, antisera from guinea pigs immunized with this PMD-generated HA show a skewed immune response toward the stem, as demonstrated by a more cross-reactive Ab response compared with antisera obtained from animals immunized with unmodified HA.

Discussion Our results demonstrate that PMD can serve as a generalizable and potentially high-resolution immunofocusing strategy that can be widely combined with other immunofocusing methods. With HEWL, we show that the PMD protocol keeps the epitope of interest intact while decreasing antigenicity elsewhere on the protein. With HA, we show that a PMD-generated antigen provides greatly reduced mAb binding at the head region while retaining robust binding to the stem region. With PMD-HA used as bait in FACS experiments, yeast clones expressing scFvs that bind to the stem region of HA were selectively enriched from a mini-library. Finally, when this PMD-HA antigen was used to immunize guinea pigs, the resultant antiserum was more cross-reactive to HAs from other influenza strains compared with animals immunized with unmodified HA. Although the in vivo derived effects are modest, taken together, our experiments demonstrate the viability of PMD for use in immunogen design. Possible immediate steps to improve the efficacy of H1+9+PEG as an immunogen include (i) introducing additional lysine substitution(s) to eliminate the hole mapped by H2897 on the HA head, (ii) altering the PEG length or modifying reagent, and/or (iii) using other chemistries outside of NHS-esters (48). It will also be important to discover new holes that need to be eliminated with additional mAbs (e.g., with yeast-display scFv libraries). We imagine such improvements to be iterative, where new PMD candidates can be sequentially screened in vitro as outlined above before use in vivo. We also note that PMD vaccine candidates can be prioritized based on human B cell binding experiments (e.g., refs. 62 and 63). Importantly, the PMD strategy is generalizable. It requires an antigen of interest and a mAb with an epitope against which to direct a vaccine-induced Ab response. 3D structural information is helpful but not absolutely required. Generating PMD antigens with a binding partner that is not an mAb is also conceivable, for example, using cell surface receptors such as CD4 for HIV-1 (64) or SR-B1 for HCV (65). Indeed, there are many attractive targets for the application of PMD. We anticipate that another advantage of PMD is its potential to produce high-resolution epitope-focused vaccines. This is because individual residues on an antigen either are or are not protected from chemical modification during PMD. Consequently, in theory, PMD could be used to create immunofocusing antigens at individual residue resolution. For example, it is conceivable that PMD could lead to vaccine candidates that avoid eliciting nonneutralizing Abs that bind to epitopes overlapping with those of neutralizing Abs (e.g., refs. 66⇓–68). Among the many possible applications of PMD, HIV-1 is particularly interesting to consider. The initial, immunodominant Ab responses to HIV-1 are strain-specific (69⇓–71). While rare, bnAbs have been isolated from infected subjects and can be mapped to a few epitopes on HIV-1 Env (72, 73). The sequences of these bnAbs indicate that an extensive degree of somatic hypermutation generally occurs during years of viral and host coevolution (74, 75). PMD offers the possibility of creating immunogens to determine if it is possible to elicit a bnAb-like response in the absence of extensive somatic hypermutation, if other strain-specific Ab responses against HIV-1 are avoided. Today, most vaccines are produced using methods developed many decades ago. Although in some cases these have had tremendous success, most notably the eradication of smallpox, they have failed to address some of greatest medical needs in the field of vaccinology, such as HIV-1 and influenza. Modern immunofocusing methods and the discovery of bnAbs have reignited the field to target such historically intractable diseases. Since PMD uses these bnAbs and can be used in combination with other immunofocusing strategies, we hope that it will aid in creating new vaccines.

Materials and Methods More detailed information is provided in SI Appendix, Materials and Methods. Protein Expression and Purification. We expressed all proteins except lysozyme (purchased from Alfa Aesar) in Expi293F cells and purified them using NiNTA (HAs) or protein A (Abs), followed by FPLC in some cases. PMD. PMD was conducted by binding an antigen to an mAb resin (HyHEL10 for lysozyme or MEDI8852* for H1+9), then incubating with an NHS-PEG-me reagent, and finally eluting the PMD-modified antigen off the resin using 100 mM glycine pH 1.5 for lysozyme or 2 M KSCN pH 7.4 for H1+9+PEG. BLI Measurements. All BLI measurements were conducted using Octet Red96. All measurements were made in PBST+BSA buffer. A detailed experimental outline is provided in SI Appendix, Materials and Methods. Yeast-Binding Experiments. All yeast experiments were conducted in PBSM buffer. Yeast were incubated with 12.5 nM of each tetrameric bait (H1 WT, H1+9, or H1+9+PEG) for 15 min, washed, stained again with an anti–c-Myc Ab (Miltenyi Biotec) for 15 min, washed twice, and then flowed. Guinea Pig Immunizations. Here 50 μg of each immunogen (either H1 WT or H1+9+PEG) in 100 μL was mixed 1:1 with alum adjuvant (Thermo Fisher Scientific) and immunized on day 0 and day 20. Serum was analyzed on day 30.

Acknowledgments We thank A. E. Powell and members of the P.S.K. laboratory for helpful comments on early drafts of this manuscript; B. N. Bell, A. E. Powell, and R. Das for helpful discussions; and J. R. Cochran and J. E. Pak for access to instrumentation. This work was supported by the Virginia and D. K. Ludwig Fund for Cancer Research and the Chan Zuckerberg Biohub.

Footnotes Author contributions: P.A.W. and P.S.K. designed research; P.A.W. performed research; P.A.W. contributed new reagents/analytic tools; P.A.W. and P.S.K. analyzed data; and P.A.W. and P.S.K. wrote the paper.

Reviewers: J.D.B., Fred Hutchinson Cancer Research Center; and J.A.W., University of California, San Francisco.

The authors declare no conflict of interest.

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