HIV-1 VRC01 Germline-Targeting Immunogens Select Distinct Epitope-Specific B Cell Receptors.

Activating precursor B cell receptors of HIV-1 broadly neutralizing antibodies requires specifically designed immunogens. Here, we compared the abilities of three such germline-targeting immunogens against the VRC01-class receptors to activate the targeted B cells in transgenic mice expressing the germline VH of the VRC01 antibody but diverse mouse light chains. Immunogen-specific VRC01-like B cells were isolated at different time points after immunization, their VH and VL genes were sequenced, and the corresponding antibodies characterized. VRC01 B cell sub-populations with distinct cross-reactivity properties were activated by each immunogen, and these differences correlated with distinct biophysical and biochemical features of the germline-targeting immunogens. Our study indicates that the design of effective immunogens to activate B cell receptors leading to protective HIV-1 antibodies will require a better understanding of how the biophysical properties of the epitope and its surrounding surface on the germline-targeting immunogen influence its interaction with the available receptor variants in vivo.

Structural basis for Zika envelope domain III recognition by a germline version of a recurrent neutralizing antibody.

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs.

Walnut antigens can trigger autoantibody development in patients with pemphigus vulgaris through a "hit-and-run" mechanism.

BACKGROUND: Environmental factors, as well as genetic predisposition, are known to be critical for the development of autoimmunity. However, the environmental agents that trigger autoimmune responses have remained elusive. One possible explanation is the "hit-and-run" mechanism in which the inciting antigens that initiate autoimmune responses are not present at the time of overt autoimmune disease. OBJECTIVE: After our previous findings that some allergens can incite autoimmune responses, we investigated the potential role of environmental allergens in triggering autoantibody development in patients with an autoimmune skin disease, pemphigus vulgaris (PV). METHODS: Revertant/germline mAbs (with mutations on variable regions of heavy and light chains reverted to germline forms) of 8 anti-desmoglein (Dsg) 3 pathogenic mAbs from patients with PV were tested for reactivity against a panel of possible allergens, including insects, pollens, epithelia, fungi, and food antigens. RESULTS: All the PV germline mAbs were reactive to antigens from walnut, including the well-known allergen Jug r 2 and an uncharacterized 85-kDa protein component. Sera from patients with PV contained significantly greater levels of anti-Dsg3 autoantibodies than walnut-specific antibodies, suggesting that the autoreactive B-cell response in patients with PV might be initially triggered by walnut antigens but is subsequently driven by Dsg3. CONCLUSION: Our findings suggest that walnut antigens/allergens can initiate autoantibody development in patients with PV through a "hit-and-run" mechanism. The revertant/germline mAb approach might provide a paradigm for the etiological study of other allergic and autoimmune diseases.

Glycosylation of the core of the HIV-1 envelope subunit protein gp120 is not required for native trimer formation or viral infectivity.

The gp120 subunit of the HIV-1 envelope (Env) protein is heavily glycosylated at ∼25 glycosylation sites, of which ∼7-8 are located in the V1/V2 and V3 variable loops and the others in the remaining core gp120 region. Glycans partially shield Env from recognition by the host immune system and also are believed to be indispensable for proper folding of gp120 and for viral infectivity. Previous attempts to alter glycosylation sites in Env typically involved mutating the glycosylated asparagine residues to structurally similar glutamines or alanines. Here, we confirmed that such mutations at multiple glycosylation sites greatly diminish viral infectivity and result in significantly reduced binding to both neutralizing and non-neutralizing antibodies. Therefore, using an alternative approach, we combined evolutionary information with structure-guided design and yeast surface display to produce properly cleaved HIV-1 Env variants that lack all 15 core gp120 glycans, yet retain conformational integrity and multiple-cycle viral infectivity and bind to several broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies and a germline-reverted version of the bNAb VRC01. Our observations demonstrate that core gp120 glycans are not essential for folding, and hence their likely primary role is enabling immune evasion. We also show that our glycan removal approach is not strain restricted. Glycan-deficient Env derivatives can be used as priming immunogens because they should engage and activate a more divergent set of germlines than fully glycosylated Env. In conclusion, these results clarify the role of core gp120 glycosylation and illustrate a general method for designing glycan-free folded protein derivatives.

Antibody promiscuity: Understanding the paradigm shift in antigen recognition.

Affinity maturation is associated with reduced malleability of the paratope that optimizes an antibody to bind to the bonafide antigen with high specificity and affinity. However, it has been illustrated that mature antibodies tend to exhibit promiscuity despite acquisition of a relatively rigid binding pocket. Such an attribute is contrary to the established paradigm of specificity in antigen recognition. In this review, an explicit dissection of the underlying mechanisms fostering such versatility in mature antibodies has been done. Polyspecificity is essentially achieved by undergoing minimal structural rearrangement at the paratope complemented with plasticity in interaction with antigen. Besides, the structural invariance of the antigen across species could modulate mature antibody specificity. Polyreactivity has been well documented for germline antibodies as broad spectrum antibody repertoire amplification is primarily governed by recombination event of the genetic machinery, which is further expanded at the structural and functional level of interaction. Degenerate specificity in antigen recognition obviates the need to produce distinct antibody for every incoming epitope.

Application of germline antibody features to vaccine development, antibody discovery, antibody optimization and disease diagnosis.

Although the efficacy and commercial success of vaccines and therapeutic antibodies have been tremendous, designing and discovering new drug candidates remains a labor-, time- and cost-intensive endeavor with high risks. The main challenges of vaccine development are inducing a strong immune response in broad populations and providing effective prevention against a group of highly variable pathogens. Meanwhile, antibody discovery faces several great obstacles, especially the blindness in antibody screening and the unpredictability of the developability and druggability of antibody drugs. These challenges are largely due to poorly understanding of germline antibodies and the antibody responses to pathogen invasions. Thanks to the recent developments in high-throughput sequencing and structural biology, we have gained insight into the germline immunoglobulin (Ig) genes and germline antibodies and then the germline antibody features associated with antigens and disease manifestation. In this review, we firstly outline the broad associations between germline antibodies and antigens. Moreover, we comprehensively review the recent applications of antigen-specific germline antibody features, physicochemical properties-associated germline antibody features, and disease manifestation-associated germline antibody features on vaccine development, antibody discovery, antibody optimization, and disease diagnosis. Lastly, we discuss the bottlenecks and perspectives of current and potential applications of germline antibody features in the biotechnology field.

Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization.

Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2∗02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.

Therapeutic Antibodies to Ganglioside GD2 Evolved from Highly Selective Germline Antibodies.

Antibodies play a crucial role in host defense and are indispensable research tools, diagnostics, and therapeutics. Antibody generation involves binding of genomically encoded germline antibodies followed by somatic hypermutation and in vivo selection to obtain antibodies with high affinity and selectivity. Understanding this process is critical for developing monoclonal antibodies, designing effective vaccines, and understanding autoantibody formation. Prior studies have found that antibodies to haptens, peptides, and proteins evolve from polyspecific germline antibodies. The immunological evolution of antibodies to mammalian glycans has not been studied. Using glycan microarrays, protein microarrays, cell binding studies, and molecular modeling, we demonstrate that therapeutic antibodies to the tumor-associated ganglioside GD2 evolved from highly specific germline precursors. The results have important implications for developing vaccines and monoclonal antibodies that target carbohydrate antigens. In addition, they demonstrate an alternative pathway for antibody evolution within the immune system that is distinct from the polyspecific germline pathway.

The Antibody Germline/Maturation Hypothesis, Elicitation of Broadly Neutralizing Antibodies Against HIV-1 and Cord Blood IgM Repertoires.

We have previously observed that all known potent broadly neutralizing antibodies (bnAbs) against HIV-1 are highly divergent from their putative germline predecessors in contrast to bnAbs against viruses causing acute infections such as henipaviruses and SARS CoV, which are much less divergent from their germline counterparts. Consequently, we have hypothesized that germline antibodies may not bind to the HIV-1 envelope glycoprotein (Env) because they are so different compared to the highly somatically mutated HIV-1-specific bnAbs. We have further hypothesized that the immunogenicity of highly conserved epitopes on the HIV-1 envelope glycoproteins (Envs) may be reduced or eliminated by their very weak or absent interactions with germline antibodies and immune responses leading to the elicitation of bnAbs may not be initiated and/or sustained. Even if such responses are initiated, the maturation pathways are so extraordinarily complex that prolonged periods of time may be required for elicitation of bnAbs with defined unique sequences. We provided the initial evidence supporting this antibody germline/maturation hypothesis, which prompted a number of studies to design vaccine immunogens that could bind putative germline predecessors of known bnAbs and to explore complex B cell lineages. However, guiding the immune system through the exceptionally complex antibody maturation pathways to elicit known bnAbs remains a major challenge. Here, we discuss studies exploring the antibody germline/maturation hypothesis as related to elicitation of bnAbs against HIV-1 and present our recent data demonstrating the existence of germline-like precursors of VRC01 antibodies in a human cord blood IgM library.