Mapping of a Novel H3-Specific Broadly Neutralizing Monoclonal Antibody Targeting the Hemagglutinin Globular Head Isolated from an Elite Influenza Virus-Immunized Donor Exhibiting Serological Breadth.

The discovery of potent and broadly protective influenza virus epitopes could lead to improved vaccines that are resistant to antigenic drift. Here, we describe human antibody C585, isolated from a vaccinee with remarkable serological breadth as measured by hemagglutinin inhibition (HAI). C585 binds and neutralizes multiple H3N2 strains isolated between 1968 and 2016, including strains that emerged up to 4 years after B cells were isolated from the vaccinated donor. The crystal structure of C585 Fab in complex with the HA from A/Switzerland/9715293/2013 (H3N2) shows that the antibody binds to a novel and well-conserved epitope on the globular head of H3 HA and that it differs from other antibodies not only in its epitope but in its binding geometry and hypermutated framework 3 region, thereby explaining its breadth and ability to mediate hemagglutination inhibition across decades of H3N2 strains. The existence of epitopes such as the one elucidated by C585 has implications for rational vaccine design.IMPORTANCE Influenza viruses escape immunity through continuous antigenic changes that occur predominantly on the viral hemagglutinin (HA). Induction of broadly neutralizing antibodies (bnAbs) targeting conserved epitopes following vaccination is a goal of universal influenza vaccines and advantageous in protecting hosts against virus evolution and antigenic drift. To date, most of the discovered bnAbs bind either to conserved sites in the stem region or to the sialic acid-binding pocket. Generally, antibodies targeting the stem region offer broader breadth with low potency, while antibodies targeting the sialic acid-binding pocket cover narrower breadth but usually have higher potency. In this study, we identified a novel neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against a broad range of H3N2 with high potency. This epitope may provide insights for future universal vaccine design.

A broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain III.

Dengue is the most widespread vector-borne viral disease caused by dengue virus (DENV) for which there are no safe, effective drugs approved for clinical use. Here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the DENV envelop protein domain III (DIII) combined with depletion by an entry defective DIII mutant, we identified a cross-reactive human monoclonal antibody (mAb), m366.6, which bound with high affinity to DENV DIII from all four DENV serotypes. Immunogenetic analysis indicated that m366.6 is a germline-like mAb with very few somatic mutations from the closest VH and Vλ germline genes. Importantly, we demonstrated that it potently neutralized DENV both in vitro and in the mouse models of DENV infection without detectable antibody-dependent enhancement (ADE) effect. The epitope of m366.6 was mapped to the highly conserved regions on DIII, which may guide the design of effective dengue vaccine immunogens. Furthermore, as the first germline-like mAb derived from a naïve antibody library that could neutralize all four DENV serotypes, the m366.6 can be a tool for exploring mechanisms of DENV infection, and is a promising therapeutic candidate.

Rapid Elimination of Broadly Neutralizing Antibodies Correlates with Treatment Failure in the Acute Phase of Simian-Human Immunodeficiency Virus Infection.

Early human immunodeficiency virus type 1 (HIV-1) treatment during the acute period of infection can significantly limit the seeding of viral reservoirs and modify the course of disease. However, while a number of HIV-1 broadly neutralizing antibodies (bnAbs) have demonstrated remarkable efficacy as prophylaxis in macaques chronically infected with simian-human immunodeficiency virus (SHIV), intriguingly, their inhibitory effects were largely attenuated in the acute period of SHIV infection. To investigate the mechanism for the disparate performance of bnAbs in different periods of SHIV infection, we used LSEVh-LS-F, a bispecific bnAb targeting the CD4 binding site and CD4-induced epitopes, as a representative bnAb and assessed its potential therapeutic benefit in controlling virus replication in acutely or chronically SHIV-infected macaques. We found that a single infusion of LSEVh-LS-F resulted in rapid decline of plasma viral loads to undetectable levels without emergence of viral resistance in the chronically infected macaques. In contrast, the inhibitory effect was robust but transient in the acutely infected macaques, despite the fact that all macaques had comparable plasma viral loads initially. Infusing multiple doses of LSEVh-LS-F did not extend its inhibitory duration. Furthermore, the pharmacokinetics of the infused LSEVh-LS-F in the acutely SHIV-infected macaques significantly differed from that in the uninfected or chronically infected macaques. Host SHIV-specific immune responses may play a role in the viremia-dependent pharmacokinetics. Our results highlight the correlation between the fast clearance of infused bnAbs and the treatment failure in the acute period of SHIV infection and may have important implications for the therapeutic use of bnAbs to treat acute HIV infections.IMPORTANCE Currently, there is no bnAb-based monotherapy that has been reported to clear the virus in the acute SHIV infection period. Since early HIV treatment is considered critical to restricting the establishment of viral reservoirs, investigation into the mechanism for treatment failure in acutely infected macaques would be important for the therapeutic use of bnAbs and eventually towards the functional cure of HIV/AIDS. Here we report the comparative study of the therapeutic efficacy of a bnAb in acutely and chronically SHIV-infected macaques. This study revealed the correlation between the fast clearance of infused bnAbs and treatment failure during the acute period of infection.

Engineered Fc based antibody domains and fragments as novel scaffolds.

Therapeutic monoclonal antibodies (mAbs) have been successful for the therapy of a number of diseases mostly cancer and immune disorders. However, the vast majority of mAbs approved for clinical use are full size, typically in IgG1 format. These mAbs may exhibit relatively poor tissue penetration and restricted epitope access due to their large size. A promising solution to this fundamental limitation is the engineering of smaller scaffolds based on the IgG1 Fc region. These scaffolds can be used for the generation of libraries of mutants from which high-affinity binders can be selected. Comprised of the CH2 and CH3 domains, the Fc region is important not only for the antibody effector function but also for its long half-life. This review focuses on engineered Fc based antibody fragments and domains including native (dimeric) Fc and monomeric Fc as well as CH2 and monomeric CH3, and their use as novel scaffolds and binders. The Fc based binders are promising candidate therapeutics with optimized half-life, enhanced tissue penetration and access to sterically restricted binding sites resulting in an increased therapeutic efficacy. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Exceptionally potent and broadly cross-reactive, bispecific multivalent HIV-1 inhibitors based on single human CD4 and antibody domains.

Soluble forms of the human immunodeficiency virus type 1 (HIV-1) primary receptor CD4 (soluble CD4 [sCD4]) have been extensively characterized for a quarter of a century as promising HIV-1 inhibitors, but they have not been clinically successful. By combining a protein cavity-filling strategy and the power of library technology, we identified an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties, including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor, an antibody domain (m36.4) that targets the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested, with potencies about 10-, 50-, and 200-fold higher than those of the broadly neutralizing antibody VRC01, the U.S. FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity and a lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be useful for HIV-1 prevention and therapy, including eradication of the virus.

Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies.

The recently discovered Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans, with high mortality. Specific, highly effective therapeutics and vaccines against the MERS-CoV are urgently needed to save human lives and address the pandemic concerns. We identified three human monoclonal antibodies (MAbs), m336, m337, and m338, targeting the receptor (CD26/DPP4) binding domain (RBD) of the MERS-CoV spike glycoprotein from a very large naïve-antibody library (containing ∼10(11) antibodies). They bound with high affinity: equilibrium dissociation constants for the three MAbs were equal to 4.2, 9.3, and 15 nM, respectively, as measured by Biacore for Fabs binding to RBD. The avidity for IgG1 m336, m337, and m338 was even higher: 99, 820, and 560 pM, respectively. The antibodies bound to overlapping epitopes that overlap the receptor binding site on the RBD as suggested by competition experiments and further supported by site-directed mutagenesis of the RBD and a docking model of the m336-RBD complex. The highest-affinity MAb, m336, neutralized both pseudotyped and live MERS-CoV with exceptional potency, 50% neutralization at 0.005 and 0.07 µg/ml, respectively, likely by competing with DPP4 for binding to the S glycoprotein. The exceptionally high neutralization activity of these antibodies and especially m336 suggests that they have great potential for prophylaxis and therapy of MERS-CoV infection in humans and as a tool for development of vaccine immunogens. The rapid identification (within several weeks) of potent MAbs suggests a possibility to use the new large antibody library and related methodology for a quick response to the public threat resulting from emerging coronaviruses. Importance: A novel human coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), was found to infect humans with a high mortality rate in 2012, just 1 decade after the appearance of the first highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). There are no effective therapeutics available. It is highly desirable to find an approach for rapidly developing potent therapeutics against MERS-CoV, which not only can be implemented for MERS treatment but also can help to develop a platform strategy to combat future emerging coronaviruses. We report here the identification of human monoclonal antibodies (MAbs) from a large nonimmune antibody library that target MERS-CoV. One of the antibodies, m336, neutralized the virus with exceptional potency. It therefore may have great potential as a candidate therapeutic and as a reagent to facilitate the development of vaccines against MERS-CoV.

N-terminal truncation of an isolated human IgG1 CH2 domain significantly increases its stability and aggregation resistance.

Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions, and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that the removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5 °C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity, and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak binding of CH2 to shFcRn as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins.

Deep sequencing and Circos analyses of antibody libraries reveal antigen-driven selection of Ig VH genes during HIV-1 infection.

The vast diversity of antibody repertoires is largely attributed to heavy chain (V(H)) recombination of variable (V), diversity (D) and joining (J) gene segments. We used 454 sequencing information of the variable domains of the antibody heavy chain repertoires from neonates, normal adults and an HIV-1-infected individual, to analyze, with Circos software, the VDJ pairing patterns at birth, adulthood and a time-dependent response to HIV-1 infection. Our comparative analyses of the Ig VDJ repertoires from these libraries indicated that, from birth to adulthood, VDJ recombination patterns remain the same with some slight changes, whereas some V(H) families are selected and preferentially expressed after long-term infection with HIV-1. We also demonstrated that the immune system responds to HIV-1 chronic infection by selectively expanding certain HV families in an attempt to combat infection. Our findings may have implications for understanding immune responses in pathology as well as for development of new therapeutics and vaccines.

Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations.

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations.

Characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: Implications for mechanisms of immune evasion and design of vaccine immunogens.

We have previously observed that all known HIV-1 broadly neutralizing antibodies (bnAbs) are highly divergent from germline antibodies in contrast to bnAbs against Hendra virus, Nipah virus and SARS coronavirus (SARS CoV). We have hypothesized that because the germline antibodies are so different from the mature HIV-1-specific bnAbs they may not bind the epitopes of the mature antibodies and provided the first evidence to support this hypothesis by using individual putative germline-like predecessor antibodies. To further validate the hypothesis and understand initial immune responses to different viruses, two phage-displayed human cord blood-derived IgM libraries were constructed which contained mostly germline antibodies or antibodies with very low level of somatic hypermutations. They were panned against different HIV-1 envelope glycoproteins (Envs), SARS CoV protein receptor-binding domain (RBD), and soluble Hendra virus G protein (sG). Despite a high sequence and combinatorial diversity observed in the cord blood-derived IgM antibody repertoire, no enrichment for binders of Envs was observed in contrast to considerable specific enrichments produced with panning against RBD and sG; one of the selected monoclonal antibodies (against the RBD) was of high (nM) affinity with only few somatic mutations. These results further support and expand our initial hypothesis for fundamental differences in immune responses leading to elicitation of bnAbs against HIV-1 compared to SARS CoV and Hendra virus. HIV-1 uses a strategy to minimize or eliminate strong binding of germline antibodies to its Env; in contrast, SARS CoV and Hendra virus, and perhaps other viruses causing acute infections, can bind germline antibody or minimally somatically mutated antibodies with relatively high affinity which could be one of the reasons for the success of sG and RBD as vaccine immunogens.

Cross-reactive HIV-1-neutralizing human monoclonal antibodies identified from a patient with 2F5-like antibodies.

The genes encoding broadly HIV-1-neutralizing human monoclonal antibodies (MAbs) are highly divergent from their germ line counterparts. We have hypothesized that such high levels of somatic hypermutation could pose a challenge for elicitation of the broadly neutralizing (bn) Abs and that identification of less somatically mutated bn Abs may help in the design of effective vaccine immunogens. In a quest for such bn Abs, phage- and yeast-displayed antibody libraries, constructed using peripheral blood mononuclear cells (PBMCs) from a patient with bn serum containing Abs targeting the epitope of the bn MAb 2F5, were panned against peptides containing the 2F5 epitope and against the HIV-1 gp140(JR-FL). Two MAbs (m66 and m66.6) were identified; the more mutated variant (m66.6) exhibited higher HIV-1-neutralizing activity than m66, although it was weaker than 2F5 in a TZM-bl cell assay. Binding of both MAbs to gp41 alanine substitution mutant peptides required the DKW(664-666) core of the 2F5 epitope and two additional upstream residues (L(660,663)). The MAbs have long (21-residue) heavy-chain third complementarity-determining regions (CDR-H3s), and m66.6 (but not m66) exhibited polyspecific reactivity to self- and non-self-antigens. Both m66 and m66.6 are significantly less divergent from their germ line Ab counterparts than 2F5--they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germ line gene products compared to 25 for 2F5. These new MAbs could help explore the complex maturation pathways involved in broad neutralization and its relationship with auto- and polyreactivity and may aid design of vaccine immunogens and development of therapeutics against HIV-1 infection.

Characterization of human IgG repertoires in an acute HIV-1 infection.

All known broadly neutralizing antibodies (bnAbs) are highly somatically mutated and therefore significantly differ from their germline predecessors. Thus although the mature bnAbs bind to conserved epitopes of the HIV-1 envelope glycoprotein (Env) with high affinity their germline predecessors do not or weakly bind Envs failing to initiate an effective immune response. The identification of less somatically mutated bnAbs and/or antibody maturation intermediates that are clonally related to bnAbs may be useful to circumvent the major problem of initiating immune responses leading to elicitation of bnAbs. Here, we describe the identification of IgG antibodies from an acutely HIV-1-infected patient using a combination of phage display and high-throughput sequencing. We found two antibodies with only a single point mutation in the V region of their heavy chain variable domains compared to their putative germline predecessors which bound with high affinity to several Envs. They targeted the Env gp41 and did not neutralize HIV-1. Using high-throughput sequencing, we identified several highly abundant CDR3s, germline-like as well as somatically mutated V genes in the VH/VL repertoires of the patient which may provide antibody intermediates corresponding to known bnAbs as templates for design of novel HIV-1 vaccine immunogens.

Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.

Animal immunization merges with innovative technologies: A new paradigm shift in antibody discovery.

Animal-derived antibody sources, particularly, transgenic mice that are engineered with human immunoglobulin loci, along with advanced antibody generation technology platforms have facilitated the discoveries of human antibody therapeutics. For example, isolation of antigen-specific B cells, microfluidics, and next-generation sequencing have emerged as powerful tools for identifying and developing monoclonal antibodies (mAbs). These technologies enable not only antibody drug discovery but also lead to the understanding of B cell biology, immune mechanisms and immunogenetics of antibodies. In this perspective article, we discuss the scientific merits of animal immunization combined with advanced methods for antibody generation as compared to animal-free alternatives through in-vitro-generated antibody libraries. The knowledge gained from animal-derived antibodies concerning the recombinational diversity, somatic hypermutation patterns, and physiochemical properties is found more valuable and prerequisite for developing in vitro libraries, as well as artificial intelligence/machine learning methods to discover safe and effective mAbs.

A platform-agnostic, function first-based antibody discovery strategy using plasmid-free mammalian expression of antibodies.

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery. In this study, we describe a process to identify and rapidly re-format and express antibodies for functional characterization. We used two different approaches to isolate antibodies to five different targets: 1) flow cytometry to identify antigen-specific single B cells from the spleen of immunized human immunoglobulin transgenic mice; and 2) panning of phage libraries. PCR amplification allowed recovery of paired VH and VL sequences from 79% to 96% of antigen-specific B cells. All cognate VH and VL transcripts were formatted into transcription and translation compatible linear DNA expression cassettes (LEC) encoding whole IgG or Fab. Between 92% and 100% of paired VH and VL transcripts could be converted to LECs, and nearly 100% of them expressed as antibodies when transfected into Expi293F cells. The concentration of IgG in the cell culture supernatants ranged from 0.05 µg/ml to 145.8 µg/ml (mean = 18.4 µg/ml). Antigen-specific binding was displayed by 78-100% of antibodies. High throughput functional screening allowed the rapid identification of several functional antibodies. In summary, we describe a plasmid-free system for cloning and expressing antibodies isolated by different approaches, in any format of choice for deep functional screening that can be applied in any research setting during antibody discovery.

Influenza vaccination in the elderly boosts antibodies against conserved viral proteins and egg-produced glycans.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galß1-4GalNAcß), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

Landscape of Non-canonical Cysteines in Human VH Repertoire Revealed by Immunogenetic Analysis.

Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insights into B cell immunogenetics and paratope diversity. By analyzing large public NGS datasets, we map the landscape of non-canonical cysteines in human variable heavy-chain domains (VHs) at the repertoire level. We identify remarkable usage of non-canonical cysteines within the heavy-chain complementarity-determining region 3 (CDR-H3) and other CDRs and framework regions. Furthermore, our study reveals the diversity and location of non-canonical cysteines and their associated motifs in human VHs, which are reminiscent of and more complex than those found in other non-human species such as chicken, camel, llama, shark, and cow. These results explain how non-canonical cysteines strategically occur in the human antibodyome to expand its paratope space. This study will guide the design of human antibodies harboring disulfide-stabilized long CDR-H3s to access difficult-to-target epitopes and influence a paradigm shift in developability involving non-canonical cysteines.

Germline-like predecessors of broadly neutralizing antibodies lack measurable binding to HIV-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens.

Several human monoclonal antibodies (hmAbs) including b12, 2G12, and 2F5 exhibit relatively potent and broad HIV-1-neutralizing activity. However, their elicitation in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. We have hypothesized that HIV-1 has evolved a strategy to reduce or eliminate the immunogenicity of the highly conserved epitopes of such antibodies by using "holes" (absence or very weak binding to these epitopes of germline antibodies that is not sufficient to initiate and/or maintain an efficient immune response) in the human germline B cell receptor (BCR) repertoire. To begin to test this hypothesis we have designed germline-like antibodies corresponding most closely to b12, 2G12, and 2F5 as well as to X5, m44, and m46 which are cross-reactive but with relatively modest neutralizing activity as natively occurring antibodies due to size and/or other effects. The germline-like X5, m44, and m46 bound with relatively high affinity to all tested Envs. In contrast, germline-like b12, 2G12, and 2F5 lacked measurable binding to Envs in an ELISA assay although the corresponding mature antibodies did. These results provide initial evidence that Env structures containing conserved vulnerable epitopes may not initiate humoral responses by binding to germline antibodies. Even if such responses are initiated by very weak binding undetectable in our assay it is likely that they will be outcompeted by responses to structures containing the epitopes of X5, m44, m46, and other antibodies that bind germline BCRs with much higher affinity/avidity. This hypothesis, if further supported by data, could contribute to our understanding of how HIV-1 evades immune responses and offer new concepts for design of effective vaccine immunogens.

Structure of an isolated unglycosylated antibody C(H)2 domain.

The C(H)2 (C(H)3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein-protein interactions with another C(H)2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the C(H)2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody gamma1 C(H)2 domain was determined at 1.7 A resolution and compared with corresponding C(H)2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody C(H)2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the C(H)2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications.

ProTherm and ProNIT: thermodynamic databases for proteins and protein-nucleic acid interactions.

ProTherm and ProNIT are two thermodynamic databases that contain experimentally determined thermodynamic parameters of protein stability and protein-nucleic acid interactions, respectively. The current versions of both the databases have considerably increased the total number of entries and enhanced search interface with added new fields, improved search, display and sorting options. As on September 2005, ProTherm release 5.0 contains 17,113 entries from 771 proteins, retrieved from 1497 scientific articles (approximately 20% increase in data from the previous version). ProNIT release 2.0 contains 4900 entries from 273 research articles, representing 158 proteins. Both databases can be queried using WWW interfaces. Both quick search and advanced search are provided on this web page to facilitate easy retrieval and display of the data from these databases. ProTherm is freely available online at http://gibk26.bse.kyutech.ac.jp/jouhou/Protherm/protherm.html and ProNIT at http://gibk26.bse.kyutech.ac.jp/jouhou/pronit/pronit.html.