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Summary Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3′ interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies., Graphical abstract, Highlights • Crystal structures unveil molecular basis of SEFL2.2 and CPM technologies • Next gen structure-guided design of CPMs to steer HC-HC pairing • Top CPMs show high pairing efficiency and optimal expression and stability • Balancing CPM charge distribution minimizes impact of sequence diversity, Biochemistry; Bioengineering; Biomolecular engineering; Structural biology

Summary Epitope-targeted HIV vaccine design seeks to focus antibody responses to broadly neutralizing antibody (bnAb) sites by sequential immunization. A chimpanzee simian immunodeficiency virus (SIV) envelope (Env) shares a single bnAb site, the variable loop 2 (V2)-apex, with HIV, suggesting its possible utility in an HIV immunization strategy. Here, we generate a chimpanzee SIV Env trimer, MT145K, which displays selective binding to HIV V2-apex bnAbs and precursor versions, but no binding to other HIV specificities. We determine the structure of the MT145K trimer by cryo-EM and show that its architecture is remarkably similar to HIV Env. Immunization of an HIV V2-apex bnAb precursor Ab-expressing knockin mouse with the chimpanzee MT145K trimer induces HIV V2-specific neutralizing responses. Subsequent boosting with an HIV trimer cocktail induces responses that exhibit some virus cross-neutralization. Overall, the chimpanzee MT145K trimer behaves as expected from design both in vitro and in vivo and is an attractive potential component of a sequential immunization regimen to induce V2-apex bnAbs., Graphical Abstract, Highlights • A designed chimpanzee SIV Env trimer binds HIV V2-apex bnAbs specifically • The trimer (MT145K) is engineered to bind inferred unmutated versions of HIV V2-apex bnAbs • The cryo-EM structure of the SIV MT145K trimer closely resembles that of HIV trimers • The MT145K SIV trimer induces HIV-specific nAb responses in a favorable animal model, Design of immunogens and strategies that can induce protective broadly neutralizing antibodies (bnAbs) is a priority for HIV vaccine development. Andrabi et al. design a chimpanzee simian immunodeficiency virus (SIV) envelope trimer immunogen that binds specifically to HIV V2-apex bnAbs and their unmutated versions. The SIV trimer immunogen induces HIV-specific neutralizing antibodies (nAbs) in a favorable animal model.

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented and the impact on public health and the global economy continues to be devastating. Although early therapies such as prophylactic antibodies and vaccines show great promise, there are concerns about the long-term efficacy and universal applicability of these therapies as the virus continues to mutate. Thus, protein-based immunogens that can quickly respond to viral changes remain of continued interest. The Spike protein, the main immunogen of this virus, displays a highly dynamic trimeric structure that presents a challenge for therapeutic development. Here, guided by the structure of the Spike trimer, we rationally design new Spike constructs that show a uniquely high stability profile while simultaneously remaining locked into the immunogen-desirable prefusion state. Furthermore, our approach emphasizes the relationship between the highly conserved S2 region and structurally dynamic Receptor Binding Domains (RBD) to enable vaccine development as well as the generation of antibodies able to resist viral mutation.

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Bispecific antibodies, engineered to recognize two targets simultaneously, demonstrate exceptional clinical potential for the therapeutic intervention of complex diseases. However, these molecules are often composed of multiple polypeptide chains of differing sequences. To meet industrial scale productivity, enforcing the correct quaternary assembly of these chains is critical. Here, we describe Chain Selectivity Assessment (CSA), a high-throughput method to rationally select parental monoclonal antibodies (mAbs) to make bispecific antibodies requiring correct heavy/light chain pairing. By deploying CSA, we have successfully identified mAbs that exhibit a native preference toward cognate chain pairing that enables the production of hetero-IgGs without additional engineering. Furthermore, CSA also identified rare light chains (LCs) that permit positive binding of the non-cognate arm in the common LC hetero-IgGs, also without engineering. This rational selection of parental mAbs with favorable developability characteristics is critical to the successful development of bispecific molecules with optimal manufacturability properties.

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

VRC01-class antibodies neutralize diverse HIV-1 strains by targeting the conserved CD4-binding site. Despite extensive investigations, crucial events in the early stage of VRC01 development remain elusive. We demonstrated how VRC01-class antibodies emerged in a Chinese donor by antigen-specific single B cell sorting, structural and functional studies, and longitudinal antibody and virus repertoire analyses. A monoclonal antibody DRVIA7 with modest neutralizing breadth was isolated that displayed a subset of VRC01 signatures. X-ray and EM structures revealed a VRC01-like angle of approach, but less favorable interactions between the DRVIA7 light-chain CDR1 and the N terminus with N276 and V5 glycans of gp120. Although the DRVIA7 lineage was unable to acquire broad neutralization, longitudinal analysis revealed a repertoire-encoded VRC01 light-chain CDR3 signature and VRC01-like neutralizing heavy-chain precursors that rapidly matured within 2 years. Thus, light chain accommodation of the glycan shield should be taken into account in vaccine design targeting this conserved site of vulnerability.

HIV is responsible for millions of deaths around the world and in the absence of available treatment capable of a cure, only the vaccine can offer protection against this virus. However, and after three decades of research, such a vaccine remains elusive. Here, I attempt to explain the major challenges on the development of an anti-HIV immunogen, and how the three-dimensional pictures of antibodies interacting with this virus can guide us to the design of a successful vaccine.

Summary: Calcitonin-gene-related peptide (CGRP) plays a key role in migraine pathophysiology. Aimovig (erenumab; erenumab-aooe in the United States) is the only US Food and Drug Administration (FDA)-approved monoclonal antibody (mAb) therapy against the CGRP receptor (CGRPR) for the prevention of migraine. Aimovig is also the first FDA-approved mAb against a G-protein-coupled receptor (GPCR). Here, we report the architecture and functional attributes of erenumab critical for its potent antagonism against CGRPR. The crystal structure of erenumab in complex with CGRPR reveals a direct ligand-blocking mechanism, enabled by a remarkable 21-residue-long complementary determining region (CDR)-H3 loop, which adopts a tyrosine-rich helix-turn tip and projects into the deep interface of the calcitonin receptor-like receptor (CLR) and RAMP1 subunits of CGRPR. Furthermore, erenumab engages with residues specific to CLR and RAMP1, providing the molecular basis for its exquisite selectivity. Such structural insights reveal the drug action mechanism of erenumab and shed light on developing antibody therapeutics targeting GPCRs. : Migraine is a neurological condition that affects more than 10% of the world population, and it is related to aberrant CGRPR signaling. Garces et al. describe the molecular mechanism by which erenumab, the first human antibody approved by the FDA for migraine prevention, specifically targets CGRPR pathway, preventing migraine. Keywords: CGRPR, erenumab, migraine, monoclonal antibodies, GPCR, CDR-H3 loop, Aimovig

Stabilized HIV-1 envelope glycoproteins (Env) that resemble the native Env are utilized in vaccination strategies aimed at inducing broadly neutralizing antibodies (bNAbs). To limit the exposure of rare isolate-specific antigenic residues/determinants we generated a SOSIP trimer based on a consensus sequence of all HIV-1 group M isolates (ConM). The ConM trimer displays the epitopes of most known bNAbs and several germline bNAb precursors. The crystal structure of the ConM trimer at 3.9 Å resolution resembles that of the native Env trimer and its antigenic surface displays few rare residues. The ConM trimer elicits strong NAb responses against the autologous virus in rabbits and macaques that are significantly enhanced when it is presented on ferritin nanoparticles. The dominant NAb specificity is directed against an epitope at or close to the trimer apex. Immunogens based on consensus sequences might have utility in engineering vaccines against HIV-1 and other viruses., Stabilized, native-like trimers of the HIV envelope protein, such as SOSIP trimers, are potential antigens for an HIV vaccine. Here, the authors generate a SOSIP trimer based on the consensus sequence of group M isolates, determine its structure and exposure of common epitopes, and show immunogenicity in rabbits and non-human primates.

The Knowledge management is a fundamental and complex process for nowadays organizations. There are several factors that affect its implementation, in practice; the low motivation of people belonging to organizations imposes an interesting challenge to multiple research trends and areas. The approach addressed in this paper to encourage and facilitate people engaged in such process is termed gamification. Gamification is based on games principles. It is useful for solving engagement issues within organizations applied in processes. This paper introduces an adapted model to the conditions of software organizations allowing identification, creation, application, transferring and evaluation of knowledge, using gamification principles. Model application and obtained results are discussed and analyzed, allowing its repeatability by the reader. It was concluded that results of the presented research-in-progress are promising.

The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody–gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.

One of the main goals on this document is for obtain a wise knowledge about the diverse impacts and support the decisions in the social politics; especially in the evaluation and assignation of resources to social invest in programs or units where counting with concepts and tools that let the measurement of social values, and their different dimensions. Planning a program is an essential part to count with the effects from it to the general population. The depth and width of the evaluation on the Social Value and the activities toward the adaptation of the program to social criteria depend on the predicable charge, sensitive of offered services, the complex on the program, the available information as well the evaluation during the course. It is really important to keep a close attention to the programs especially those that create a big social impact. In order to define the framework and possible central aspects of the evaluation of social value will require sorting and examining the social effects of the program planned by their impact, scope and importance, using information commonly available and material relevant work. Based on this information, the corresponding terms of reference for the evaluation of social value will be made. This document is primarily aimed at technicians and specialists of social activity in the institutions that need to evaluate social programs.

SummaryThe HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121–123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans.

International audience; The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

Mammalian polynucleotide kinase 3’ phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the non-homologous end-joining (NHEJ) and base-excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3’-phosphates from, or by phosphorylating 5’-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3’-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end-seeking. The structure of dsDNA bound to the 5’-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA-ends in the context of single-strand and double-strand breaks, and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.

The capacity to both ferment and oxidize l -ascorbate has been widely documented for a number of enteric bacteria. Here we present evidence that all the strains of Klebsiella pneumoniae tested in this study ferment l -ascorbate using the ula regulon-encoded proteins. Under aerobic conditions, several phenotypes were observed for the strains. Our results showed that the yiaK-S system is required for this aerobic metabolic process. Gel shift experiments performed with UlaR and YiaJ and probes corresponding to the specific promoters indicated that l -ascorbate-6-phosphate is the effector molecule recognized by both regulators, since binding of the repressors to their recognition sites was impaired by the presence of this compound. We demonstrated that in K. pneumoniae cells l -ascorbate-6-phosphate is formed only by the action of the UlaABC phosphotransferase system. This finding explains why strains that lack the ula genetic system and therefore are unable to form the inducer intracellularly cannot efficiently use this vitamin as a carbon source under either anaerobic or aerobic conditions. Thus, efficient aerobic metabolism of l -ascorbate in K. pneumoniae is dependent on the presence of both the yiaK-S and ula systems. The expression of the yiaK-S operon, but not the expression of the ula regulon, is controlled by oxygen availability. Both systems are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and by IHF.

UlaR is a DNA binding protein of the DeoR family of eubacterial transcriptional repressors which maintains the utilization of the L-ascorbate ula regulon in a repressed state. The availability of L-ascorbate in the growth medium releases UlaR-mediated repression on the ula regulon, thereby activating transcription. The molecular details of this induction by L-ascorbate have remained elusive to date. Here we have identified L-ascorbate 6-phosphate as a direct effector of UlaR; using a combination of site-directed mutagenesis, gel retardation, isothermal titration calorimetry, and analytical ultracentrifugation studies, we have identified the key amino acid residues that mediate L-ascorbate 6-phosphate binding and constructed the first model of regulation of a DeoR family member, establishing the basis of the ula regulon transcription control by UlaR. In this model, specific quaternary rearrangements of the DeoR-type repressor are the molecular underpinning of the activating and repressing forms. A DNA-bound UlaR tetramer establishes repression, whereas an L-ascorbate-6-phosphate-induced breakdown of the tetrameric configuration in favor of an UlaR dimeric state results in dissociation of UlaR from DNA and allows transcription of ulaG and ulaABCDEF structural genes. Despite the fact that similar changes have been described for other unrelated repressor factors, this is the first report to demonstrate that specific oligomerization changes are responsible for the activating and repressing forms of a DeoR-type eubacterial transcriptional repressor.