Broadly neutralizing antibodies (bnAbs) against HIV-1 are promising components of long-acting antiretroviral treatment (ART) due to their prolonged activity and potential for recruiting additional immune effector functions. Their use is most advanced in virologically suppressed people living with HIV-1 switching from a standard daily ART regimen to a long-acting regimen comprising one or two bnAbs combined with one of the currently approved long-acting small-molecule inhibitors: cabotegravir or lenacapavir. Implementation of bnAb-based therapies, however, requires accurate assessment of viral susceptibility. Although phenotypic assays provide the most direct and reliable measures of bnAb activity, their limited scalability has prompted interest in genotypic approaches. Here we summarize evidence linking HIV-1 Env sequence characteristics to reduced bnAb susceptibility, including in vivo selection data in animal models and human studies, and in vitro selection and susceptibility data. Although genotypic analysis of HIV-1 Env may reliably identify resistance in some viruses, more research is required before genotypic methods can reliably predict susceptibility in clinical settings.

The Ras-Raf-MEK-ERK (MAPK) pathway is a signal transduction cascade used to regulate cellular processes including cell cycle progression and proliferation. Aberrant activation of this pathway is implicated in cancer development, and treatment with Raf, MEK and ERK inhibitors often leads to adaptive resistance. Combination therapies have been shown to offer benefits; however, the MEK-ERK interface remains poorly understood, hindering structure-guided approaches to the design of potent MAPK pathway inhibitors targeting this complex. To identify important residues for the MEK1-ERK2 interaction, we performed site-directed mutagenesis on ERK2. We used circular dichroism to assess the secondary structure of the ERK2 mutants. We also used biolayer interferometry binding experiments coupled with phosphorylation assays to evaluate the impact of the mutated residues on the formation of the MEK1-ERK2 complex and activity. Circular dichroism showed no differences in secondary structure for any of our ERK2 mutants. Of all the mutations generated, the L234D mutation in ERK abrogated binding and phosphorylation by MEK1 the most. Other mutants showed some reductions in binding or activity but require further analysis. Of note, L234 is located on an ERK2 α-helix adjacent to the phosphorylation lip, consistent with MEK1 binding this face during phosphorylation. Our results suggest that this α-helix may play critical roles in the MEK1-ERK2 complex. Studying the impact of additional mutations in this and additional regions will develop our understanding of the MEK-ERK interface and inform the design of allosteric inhibitors that can modulate MEK-ERK complex formation.

The mitogen-activated protein kinase (MAPK) pathway plays a critical role in controlling cell cycle progression and cell proliferation. This Ras-Raf-MEK-ERK pathway is constitutively activated in over 80% of melanomas, as well as in some pancreatic, colon, lung, ovarian, and kidney cancers. Anticancer drugs targeting proteins earlier in the pathway, such as B-Raf and MEK, often lose efficacy due to the development of resistance. Thus, drugs that target allosteric sites hold promise if used in combination with ATP-competitive inhibitors that have already been developed. We have been working on identifying allosteric sites on key kinases of this pathway that could be used for future drug discovery efforts. We previously found that the alpha-G helix of B-Raf is critical for its interaction with MEK and that mutations of several residues along this helix could completely abrogate binding and downstream phosphorylation activity in vitro. With this information, we further analyzed the crystal structure of the B-Raf-MEK complex to generate mutations in MEK to determine if the same region of MEK that contacts the B-Raf alpha-G helix is important for binding ERK. Additionally, since no complex structure of the MEK-ERK complex was available, we generated mutations in different regions of the C-lobe of ERK as a starting point to identify a region of contact. We tested the effects of our mutations on binding using pull downs and biolayer interferometry. We also assessed phosphorylation levels of ERK in vitro. Our MEK mutants displayed no difference in binding to B-Raf, but did have altered binding to ERK. This suggests that the modes of MEK binding to B-Raf versus ERK differ. Moreover, our mapping of the ERK interface has led to the identification of a residue on the helix spanning residues 232-245 that, when mutated, significantly reduces both binding to MEK and phosphorylation of ERK. Additional work is needed to determine if our mutation affects the conformation of a nearby alpha helix of ERK and as a result affects MEK binding or if it is due to a direct effect. Nevertheless, these results demonstrate that MEK-ERK binding and downstream activity can be altered by targeting sites outside their catalytic region.

HIV evolves very quickly, so approaches to design an effective vaccine that elicits protective antibodies have thus far been unsuccessful. Current HIV vaccine design efforts seek to elicit broadly neutralizing antibodies, unique antibodies that target many viral variants, by first eliciting their precursors through prime‐boost regimens. This requires an understanding of the co‐evolution between viruses and antibodies. Towards this goal, we are analyzing a cooperating antibody, called DH475, which exerted pressure on HIV to evolve in such a way that it became sensitive to the DH270 broadly neutralizing antibody lineage. This study aims to elucidate how DH475 binds to the HIV viral spike and identify how DH475 facilitated the development of DH270 broadly neutralizing antibodies. We obtained a 2.90Å crystal structure of DH475 in complex with Man9 glycan, and used site‐directed mutagenesis coupled with biolayer interferometry (BLI) and protein‐protein docking to characterize how DH475 interacts with Env. These investigations revealed a glycan‐dependent epitope, and docking analyses identified an unorthodox binding mode in which the DH475 framework and constant regions participate in binding. While further confirmation of DH475’s binding mode is required, our findings indicate an overlapping epitope between the DH270 lineage and DH475, consistent with its cooperative neutralization ability.

Elicitation of broadly neutralizing antibodies (bnAbs) is a goal of vaccine design as a strategy for targeting highly divergent strains of HIV-1. Current HIV-1 vaccine design efforts seek to elicit bnAbs by first eliciting their precursors through prime-boost regimens. This requires an understanding of the co-evolution between viruses and antibodies. Towards this goal, we have analyzed two cooperating antibodies, DH475 and DH272, which exerted pressure on the HIV population in an infected donor, called CH848, to evolve in such a way that it became sensitive to the V3-glycan supersite DH270 bnAb lineage. We obtained a 2.90Å crystal structure of DH475 in complex with the Man9 glycan and a negative stain EM model of DH272 in complex with the HIV-1 spike trimer, Env. Coupled with additional modeling studies and biochemical data, our studies reveal that DH475 contacts a V3- and V4-glycan dependent epitope accessible on an open or shed Env and that DH272 makes critical contacts with the V1V2 and V3 loops on HIV-1 Env. Using these data, we suggest a prime-boost regimen that may facilitate the initiation of DH270-like bnAb precursors.

The mitogen-activated protein kinase (MAPK) pathway is pertinent to various cellular processes such as cell proliferation, cell division, and cell death. Dysregulation of the MAPK pathway can therefore lead to numerous illnesses. Most notably, the dysregulation of proteins in this pathway is a significant cause of melanomas and other cancers. Due to this oncogenic potential, various drugs and inhibitors exist to regulate MAPK overexpression. As drug resistance and paradoxical activation are perpetual challenges, additional regulatory targets are needed to effectively suppress the effects of MAPK oncogenic mutations, calling for further investigation into downstream interactions such as that between MEK and ERK kinase. By identifying and characterizing the binding interface between MEK and ERK, we can identify additional drug targets that can modulate the MAPK pathway. To help map the MEK-ERK interface, we first performed computational docking experiments to find possible interaction regions between MEK and ERK. In several trials, we found that certain MEK alpha helices interacted with ERK. We then proceeded to identify the corresponding binding partners on ERK. We introduced mutations in the most promising regions on ERK and expressed the mutant proteins using a bacterial expression system. Using biolayer interferometry and co-elution assays, we compared the binding affinities of our mutants to that of the corresponding wild-type. Our studies showed that introducing the K231E and K114E mutations in ERK altered the binding kinetics between MEK and ERK. Our co-elution studies showed that a stable complex does not occur, suggesting that the interaction between MEK and ERK is either weak or transient and may need another molecule to stabilize the complex. Cross-linking experiments are in progress, and so far, we identified 1% glutaraldehyde incubated for 5 minutes as sufficient for covalent linkage of MEK and ERK. These findings will help towards obtaining a 3D MEK-ERK complex structure to better understand signaling in this pathway.

Broadly neutralizing antibodies (bnAbs) are promising for HIV-1 vaccine design because they target highly divergent strains of the virus. However, attempts to elicit bnAbs have not been successful because of their long maturation pathways and high mutation frequencies. Thus, the design of an effective vaccine to elicit bnAbs will require an understanding of the co-evolution process between the virus and antibodies in a host. In one HIV-1 infected patient called CH848, the elicitation of a bnAb lineage called DH270 was preceded by a cooperating antibody lineage, of which DH272 was a member. This antibody was observed to neutralize, or prevent infectivity, of the initial infecting virus, called the transmitted founder (TF). We characterized an antibody antigen binding fragment (Fab) closely related to DH272, called F7-22, and its interactions with the TF spike protein, called Env, from patient CH848 using negative stain electron microscopy, molecular modeling, and binding kinetics. Data implicate the Fab CDRH3 and CDRL1 loops and the Env V1/V2 loops as important for complex formation. Further work is needed to develop a more complete model of this interaction and a rationale for the mechanism through which DH272 acts as a cooperating lineage.

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-Raf V600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-Raf V600E . We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-Raf V600E -induced melanomas.

Antibodies that can neutralize diverse HIV-1 strains develop in ~10–20% of HIV-1 infected individuals, and their elicitation is a goal of vaccine design. Such antibodies can also serve as therapeutics for those who have already been infected with the virus. Structural characterizations of broadly reactive antibodies in complex with the HIV-1 spike indicate that there are a limited number of sites of vulnerability on the spike. Analysis of their structures can help reveal commonalities that would be useful in vaccine design and provide insights on combinations of antibodies that can be used to minimize the incidence of viral resistance mutations. In this review, we give an update on recent structures determined of the spike in complex with broadly neutralizing antibodies in the context of all epitopes on the HIV-1 spike identified to date.

The past several months have witnessed the emergence of SARS-CoV-2 variants with novel spike protein mutations that are influencing the epidemiological and clinical aspects of the COVID-19 pandemic. These variants can increase rates of virus transmission and/or increase the risk of reinfection and reduce the protection afforded by neutralizing monoclonal antibodies and vaccination. These variants can therefore enable SARS-CoV-2 to continue its spread in the face of rising population immunity while maintaining or increasing its replication fitness. The identification of four rapidly expanding virus lineages since December 2020, designated variants of concern, has ushered in a new stage of the pandemic. The four variants of concern, the Alpha variant (originally identified in the UK), the Beta variant (originally identified in South Africa), the Gamma variant (originally identified in Brazil) and the Delta variant (originally identified in India), share several mutations with one another as well as with an increasing number of other recently identified SARS-CoV-2 variants. Collectively, these SARS-CoV-2 variants complicate the COVID-19 research agenda and necessitate additional avenues of laboratory, epidemiological and clinical research.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM(+) IgD(+)CD27(+), thus suggesting that they originated from a pool of antigen-experienced IgM(+) or marginal zone B cells.

Coronavirus research has gained tremendous attention because of the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (nCoV or SARS-CoV-2). In this review, we highlight recent studies that provide atomic-resolution structural details important for the development of monoclonal antibodies (mAbs) that can be used therapeutically and prophylactically and for vaccines against SARS-CoV-2. Structural studies with SARS-CoV-2 neutralizing mAbs have revealed a diverse set of binding modes on the spike's receptor-binding domain and N-terminal domain and highlight alternative targets on the spike. We consider this structural work together with mAb effects in vivo to suggest correlations between structure and clinical applications. We also place mAbs against severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses in the context of the SARS-CoV-2 spike to suggest features that may be desirable to design mAbs or vaccines capable of conferring broad protection.

SummaryThe HIV-1 envelope (Env) is comprised by mass of over 50% glycans. A goal of HIV-1 vaccine development is the induction of Env glycan-reactive broadly neutralizing antibodies (bnAbs). The 2G12 bnAb recognizes an Env glycan cluster using a unique variable heavy (VH) domain-swapped conformation that results in fragment antigen-binding (Fab) dimerization. Here we describe Fab-dimerized glycan (FDG)-reactive antibodies without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques that neutralized heterologous HIV-1 isolates. FDG precursors were boosted by vaccination in macaques, and were present in HIV-1-naïve humans with an average estimated frequency of one per 340,000 B cells. These data demonstrate frequent HIV-1 Env glycan-reactive bnAb B cell precursors in macaques and humans and reveal a novel strategy for their induction by vaccination.HighlightsDiscovery of Fab-dimerized HIV-1 glycan-reactive antibodies with a non-domain-swapped architectureFab-dimerized antibodies neutralize heterologous HIV-1 isolates.Antibodies with this architecture can be elicited by vaccination in macaques.Fab-dimerized antibodies are found in HIV-1 naïve humans.

Understanding affinity maturation of antibodies that can target many variants of HIV-1 is important for vaccine development. While the antigen-binding site of antibodies is known to mutate throughout the co-evolution of antibodies and viruses in infected individuals, the roles of the mutations in the antibody framework region are not well understood. Throughout affinity maturation, the CH103 broadly neutralizing antibody lineage, from an individual designated CH505, altered the orientation of one of its antibody variable domains. The change in orientation was a response to insertions in the variable loop 5 (V5) of the HIV envelope. In this study, we generated CH103 lineage antibody variants in which residues in the variable domain interface were mutated, and measured the binding to both autologous and heterologous HIV-1 envelopes. Our data show that very few mutations in an early intermediate antibody of the lineage can improve binding toward both autologous and heterologous HIV-1 envelopes. We also crystallized an antibody mutant to show that framework mutations alone can result in a shift in relative orientations of the variable domains. Taken together, our results demonstrate the functional importance of residues located outside the antigen-binding site in affinity maturation.

Antigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The broad protection afforded by a "universal" influenza vaccine may come from immunogens that elicit humoral immune responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor-binding site (RBS). Here, we engineered candidate immunogens that use noncirculating, avian influenza HAs as molecular scaffolds to present the broadly neutralizing RBS epitope from historical, circulating H1 influenzas. These "resurfaced" HAs (rsHAs) remove epitopes potentially targeted by strain-specific responses in immune-experienced individuals. Through structure-guided optimization, we improved two antigenically different scaffolds to bind a diverse panel of pan-H1 and H1/H3 cross-reactive bnAbs with high affinity. Subsequent serological and single germinal center B cell analyses from murine prime-boost immunizations show that the rsHAs are both immunogenic and can augment the quality of elicited RBS-directed antibodies. Our structure-guided, RBS grafting approach provides candidate immunogens for selectively presenting a conserved viral epitope.

HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide (“Man 9 -V3”) for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage (“DH270”), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man 9 -V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs—the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.

Human immunodeficiency virus type 1 (HIV-1) is a rapidly evolving pathogen that causes acquired immunodeficiency syndrome (AIDS) in humans. There are ∼30-35 million people infected with HIV around the world, and ∼25 million have died since the first reported cases in 1981. In addition, each year 2-3 million people become newly infected, and >1 million die of AIDS. An HIV-1 vaccine would help halt an AIDS pandemic, and efforts to develop a vaccine have focused on targeting the HIV-1 envelope, Env, found on the surface of the virus. A number of chronically infected individuals have been shown to produce antibodies, called broadly neutralizing antibodies (bnAbs), that target many strains of HIV-1 by binding to Env, thus suggesting promise for HIV-1 vaccine development. BnAbs take years to develop, and have a number of traits that inhibit their production; thus, a number of researchers are trying to understand the pathways that result in bnAb production, so that they can be elicited more rapidly by vaccination. This review discusses results and implications from two HIV-1-infected individuals studied longitudinally who produced bnAbs against two different sites on HIV-1 Env, and immunization studies that used Envs derived from those individuals.

Non-neutralizing antibodies (nnAbs) to HIV-1 show little measurable activity in prevention or therapy in animal models yet were the only correlate of protection in the RV144 vaccine trial. To investigate the role of nnAbs on HIV-1 infection in vivo, we devised a replication-competent HIV-1 reporter virus that expresses a heterologous HA-tag on the surface of infected cells and virions. Anti-HA antibodies bind to, but do not neutralize, the reporter virus in vitro. However, anti-HA protects against infection in humanized mice and strongly selects for nnAb-resistant viruses in an entirely Fc-dependent manner. Similar results were also obtained with tier 2 HIV-1 viruses using a human anti-gp41 nnAb, 246D. While nnAbs are demonstrably less effective than broadly neutralizing antibodies (bNAbs) against HIV-1 in vitro and in vivo, the data show that nnAbs can protect against and alter the course of HIV-1 infection in vivo. PAPERCLIP.

A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env(+) B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env-upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env+ (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools.

A preventive HIV-1 vaccine should induce HIV-1-specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.