Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice.

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus.

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells, the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical, and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden.

Validation of Ligand Tetramers for the Detection of Antigen-Specific Lymphocytes.

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.

Correction: HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

[This corrects the article DOI: 10.1371/journal.ppat.1007431.].

HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.

Examining Practice and Learning Effects With Serial Administration of the Clinical Reaction Time Test in Healthy Young Athletes.

CONTEXT: The clinical reaction time (RTclin) test has been recommended as a valid test for assessing concussion and determining recovery of reaction time function following concussion. However, it is unknown whether repeat assessment, as is used in postconcussion testing, is affected by learning or practice phenomena. OBJECTIVE: To determine if a practice or learning effect is present with serial administration of the RTclin test. DESIGN: Randomized control trial. SETTING: University athletic training clinics. PARTICIPANTS: A total of 112 healthy collegiate athletes (age = 19.46 [1.34] y). INTERVENTIONS: The control group completed the RTclin test on days 1 and 60. The experimental group completed the RTclin test on days 1, 2, 3, 7, and 60. MAIN OUTCOME MEASURE: Reaction time as measured with the RTclin test. RESULTS: The difference in RTclin test performance from day 1 to day 60 was not significant (mean change = -2.77 [14.46] ms, P = .42, 95% confidence intervals, -6.40 to 0.862) between groups. The experimental group experienced significant improvement (λ = 0.784, F4,49 = 3.365, P = .02, η2 = .216, power = 0.81) with acute repeat testing. However, post hoc analysis did not reveal a significant difference between scores during the 5 test periods. CONCLUSIONS: The results suggest serial administration of the RTclin test does not produce a practice or learning effect. Clinicians, however, should be cautious as the results do provide evidence patients may demonstrate improved scores when testing occurs on repetitive days after initial exposure to the test.

Cross-protective antibodies against common endemic respiratory viruses.

Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) can cause severe disease and death in immunocompromised patients, the elderly, and those with underlying lung disease. A protective monoclonal antibody exists for RSV, but clinical use is limited to high-risk infant populations. Hence, therapeutic options for these viruses in vulnerable patient populations are currently limited. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3 × 1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens at 3.62 Å resolution for 3 × 1 bound to HPIV3 and at 2.24 Å for MxR bound to RSV, providing a structural basis for in vitro binding and neutralization. Together, a cocktail of 3 × 1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals.

Adjuvants influence the maturation of VRC01-like antibodies during immunization.

Once naive B cells expressing germline VRC01-class B cell receptors become activated by germline-targeting immunogens, they enter germinal centers and undergo affinity maturation. Booster immunizations with heterologous Envs are required for the full maturation of VRC01-class antibodies. Here, we examined whether and how three adjuvants, Poly(I:C), GLA-LSQ, or Rehydragel, that activate different pathways of the innate immune system, influence the rate and type of somatic mutations accumulated by VRC01-class BCRs that become activated by the germline-targeting 426c.Mod.Core immunogen and the heterologous HxB2.WT.Core booster immunogen. We report that although the adjuvant used had no influence on the durability of plasma antibody responses after the prime, it influenced the plasma VRC01 antibody titers after the boost and the accumulation of somatic mutations on the elicited VRC01 antibodies.

Characterization of a vaccine-elicited human antibody with sequence homology to VRC01-class antibodies that binds the C1C2 gp120 domain.

Broadly HIV-1-neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02-derived heavy chains paired with light chains expressing five-amino acid-long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line-targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans.

Development of a VRC01-class germline targeting immunogen derived from anti-idiotypic antibodies.

An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs). Broad and potent VRC01-class bNAbs have been isolated from multiple infected individuals, suggesting that they could be reproducibly elicited by vaccination. Several HIV-1 envelope-derived germline-targeting immunogens have been designed to engage naive VRC01-class precursor B cells. However, they also present off-target epitopes that could hinder development of VRC01-class bNAbs. We characterize a panel of anti-idiotypic monoclonal antibodies (ai-mAbs) raised against inferred-germline (iGL) VRC01-class antibodies. By leveraging binding, structural, and B cell sorting data, we engineered a bispecific molecule derived from two ai-mAbs; one specific for VRC01-class heavy chains and one specific for VRC01-class light chains. The bispecific molecule preferentially activates iGL-VRC01 B cells in vitro and induces specific antibody responses in a murine adoptive transfer model with a diverse polyclonal B cell repertoire. This molecule represents an alternative non-envelope-derived germline-targeting immunogen that can selectively activate VRC01-class precursors in vivo.

Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes.

Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12). We determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb12. Immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine B cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. Together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies.

Anti-idiotypic antibodies elicit anti-HIV-1-specific B cell responses.

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5-amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)-specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1.

Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core.

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1-3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.

RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays.

RNA interference (RNAi) is a process in which double-strand RNA (dsRNA) directs the specific degradation of a corresponding target mRNA. The mediators of this process are small dsRNAs, of approximately 21 bp in length, called small interfering RNAs (siRNAs). siRNAs, which can be prepared in vitro in a number of ways and then transfected into cells, can direct the degradation of corresponding mRNAs inside these cells. Hence, siRNAs represent a powerful tool for studying gene functions, as well as having the potential of being highly specific pharmaceutical agents. Some limitations in using this technology exist because the preparation of siRNA in vitro and screening for siRNAs efficient in RNAi can be expensive and time-consuming processes. Here, we demonstrate that custom oligonucleotide arrays can be efficiently used for the preparation of defined mixtures of siRNAs for the silencing of exogenous and endogenous genes. The method is fast, inexpensive, does not require siRNA optimization and has a number of advantages over methods utilizing enzymatic preparation of siRNAs by digestion of longer dsRNAs, as well as methods based on chemical synthesis of individual siRNAs or their DNA templates.

Structural basis for germline antibody recognition of HIV-1 immunogens.

Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1-infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb-426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01-class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01-class bNAbs and guidelines for structure-based immunogen design.

Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

Werner syndrome protein 1367 variants and disposition towards coronary artery disease in Caucasian patients.

The leading causes of death for individuals with Werner syndrome (WS) are myocardial infarction (MI) and stroke. The WS gene encodes a nuclear protein with both helicase and exonuclease activities. While individuals with WS have mutations that result in truncated, inactive proteins, several sequence variants have been described in apparently unaffected individuals. Some of these gene polymorphisms encode non-conservative amino acid substitutions, and it is expected that the changes would affect enzyme activity, although this has not been determined. Two research groups have studied the Cys/Arg 1367 polymorphism (located near the nuclear localization signal) in healthy and MI patients. Their results suggest that the Arg allele is protective against MI. We have characterized the Cys (C) and Arg (R) forms of the protein and find no notable difference in helicase and nuclease activities, or in nuclear/cytoplasmic distribution. The frequency of the C/R alleles in healthy individuals and subjects with coronary artery disease (CAD) drawn from the Baltimore Longitudinal Study of Aging (BLSA) was also examined. There was no indication that the R allele was protective against CAD. We conclude that the C/R polymorphism does not affect enzyme function or localization and does not influence CAD incidence in the BLSA cohort.

Physical and functional mapping of the replication protein a interaction domain of the werner and bloom syndrome helicases.

The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN(H-R)) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN(H-R) helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN(H-R) had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM(642-1290), that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.