Oleksandr Kalyuzhniy

Rational HIV Immunogen Design to Target Specific Germline B Cell Receptors

Building Better Vaccines In the past few years, several highly potent, broadly neutralizing antibodies (bNAbs) specific for the gp120 envelope protein of HIV-1 have been discovered. The goal of this work is to use this information to inform the design of vaccines that are able to induce such antibodies (see the Perspective by Crowe). However, because of extensive somatic hypermutation, the epitope bound by these antibodies often does not bind to the germline sequence. Jardine et al. (p. 711, published online 28 March; see the cover) used computational analysis and in vitro screening to design an immunogen that could bind to VRC01-class bNAbs and to their germline precursors. Georgiev et al. (p. 751) took advantage of the fact that only four sites on the HIV viral envelope protein seem to bind bNAbs, and sera that contain particular bNAbs show characteristic patterns of neutralization. An algorithm was developed that could successfully delineate the neutralization specificity of antibodies present in polyclonal sera from HIV-infected patients. Structural knowledge of broadly neutralizing antibodies against HIV-1 guides the design of an immunogen to elicit them. Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been isolated from HIV-1–infected individuals; however, such bNAbs have not been induced by vaccination. Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided, in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to multiple VRC01-class bNAbs and germline precursors, and elucidated germline binding crystallographically. When multimerized on nanoparticles, this immunogen (eOD-GT6) activates germline and mature VRC01-class B cells. Thus, eOD-GT6 nanoparticles have promise as a vaccine prime. In principle, germline-targeting strategies could be applied to other epitopes and pathogens.

Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo

Human species C adenovirus serotype 5 (Ad5) is the most common viral vector used in clinical studies worldwide. Ad5 vectors infect liver cells in vivo with high efficiency via a poorly defined mechanism, which involves virus binding to vitamin K-dependent blood coagulation factors. Here, we report that the major Ad5 capsid protein, hexon, binds human coagulation factor X (FX) with an affinity of 229 pM. This affinity is 40-fold stronger than the reported affinity of Ad5 fiber for the cellular receptor coxsackievirus and adenovirus receptor, CAR. Cryoelectron microscopy and single-particle image reconstruction revealed that the FX attachment site is localized to the central depression at the top of the hexon trimer. Hexon-mutated virus bearing a large insertion in hexon showed markedly reduced FX binding in vitro and failed to deliver a transgene to hepatocytes in vivo. This study describes the mechanism of FX binding to Ad5 and demonstrates the critical role of hexon for virus infection of hepatocytes in vivo.

Proof of principle for epitope-focused vaccine design

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

Computational Design of Epitope-Scaffolds Allows Induction of Antibodies Specific for a Poorly Immunogenic HIV Vaccine Epitope.

Broadly cross-reactive monoclonal antibodies define epitopes for vaccine development against HIV and other highly mutable viruses. Crystal structures are available for several such antibody-epitope complexes, but methods are needed to translate that structural information into immunogens that re-elicit similar antibodies. We describe a general computational method to design epitope-scaffolds in which contiguous structural epitopes are transplanted to scaffold proteins for conformational stabilization and immune presentation. Epitope-scaffolds designed for the poorly immunogenic but conserved HIV epitope 4E10 exhibited high epitope structural mimicry, bound with higher affinities to monoclonal antibody (mAb) 4E10 than the cognate peptide, and inhibited HIV neutralization by HIV+ sera. Rabbit immunization with an epitope-scaffold induced antibodies with structural specificity highly similar to mAb 4E10, an important advance toward elicitation of neutralizing activity. The results demonstrate that computationally designed epitope-scaffolds are valuable as structure-specific serological reagents and as immunogens to elicit antibodies with predetermined structural specificity.

Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold.

A two-segment HIV epitope grafted into a scaffold protein maintains high affinity for a broadly neutralizing antibody. The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

Baby steps toward bNAbs Some HIV-infected individuals develop heavily mutated, broadly neutralizing antibodies (bNAbs) that target HIV. Scientists aim to design vaccines that would elicit such antibodies. Jardine et al. report an important step toward this goal: They engineered an immunogen that could engage B cells from HIV-uninfected individuals that express the germline versions of the immunoglobulin genes harbored by a particular class of bNAbs. The frequencies of these B cells, their affinities for the immunogen, and structural analysis suggest that the immunogen is a promising candidate. Further shaping of the B cell response with subsequent immunogens may eventually elicit bNAbs in people. Science, this issue p. 1458 People that have not been infected with HIV can harbor HIV-1 broadly neutralizing antibody B cell precursors. Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Summary Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

Summary Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.

Coagulation Factor X Activates Innate Immunity to Human Species C Adenovirus

Wound Healing and Immunity Although wound healing and infection are often overlapping processes, whether the wound healing response modulates the immune response is not well understood. Doronin et al. (p. 795, published online 27 September; see the Perspective by Herzog and Ostrov) now show that coagulation factor X, an important component of the blood clotting cascade, helps to trigger antiviral immunity in response to adenovirus infection in mice. Factor X binds to human type C adenovirus with very high affinity. Structural analysis identified the critical binding residues between factor X and adenovirus, which, when mutated, inhibited binding. Despite being able to infect splenic macrophages in mice, transcriptional profiling of spleens from mice infected with a mutant adenovirus unable to bind to factor X revealed impaired activation of signaling cascades associated with innate immunity. Tagging adenovirus with a serum protein prompts an immune response when the virus enters cells. Although coagulation factors play a role in host defense for “living fossils” such as horseshoe crabs, the role of the coagulation system in immunity in higher organisms remains unclear. We modeled the interface of human species C adenovirus (HAdv) interaction with coagulation factor X (FX) and introduced a mutation that abrogated formation of the HAdv-FX complex. In vivo genome-wide transcriptional profiling revealed that FX-binding–ablated virus failed to activate a distinct network of nuclear factor κB–dependent early-response genes that are activated by HAdv-FX complex downstream of TLR4/MyD88/TRIF/TRAF6 signaling. Our study implicates host factor “decoration” of the virus as a mechanism to trigger an innate immune sensor that responds to a misplacement of coagulation FX from the blood into intracellular macrophage compartments upon virus entry into the cell.

Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob

ABSTRACT Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a KD (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.