Structural Determination of the Broadly Reactive Anti-IGHV1-69 Anti-idiotypic Antibody G6 and Its Idiotope.

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine.

A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve.

Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of 'universal' influenza vaccine strategies.

Humanized mouse G6 anti-idiotypic monoclonal antibody has therapeutic potential against IGHV1-69 germline gene-based B-CLL.

In 10-20% of the cases of chronic lymphocytic leukemia of B-cell phenotype (B-CLL), the IGHV1-69 germline is utilized as VH gene of the B cell receptor (BCR). Mouse G6 (MuG6) is an anti-idiotypic monoclonal antibody discovered in a screen against rheumatoid factors (RFs) that binds with high affinity to an idiotope expressed on the 51p1 alleles of IGHV1-69 germline gene encoded antibodies (G6-id(+)). The finding that unmutated IGHV1-69 encoded BCRs are frequently expressed on B-CLL cells provides an opportunity for anti-idiotype monoclonal antibody immunotherapy. In this study, we first showed that MuG6 can deplete B cells encoding IGHV1-69 BCRs using a novel humanized GTL mouse model. Next, we humanized MuG6 and demonstrated that the humanized antibodies (HuG6s), especially HuG6.3, displayed ∼2-fold higher binding affinity for G6-id(+) antibody compared to the parental MuG6. Additional studies showed that HuG6.3 was able to kill G6-id(+) BCR expressing cells and patient B-CLL cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Finally, both MuG6 and HuG6.3 mediate in vivo depletion of B-CLL cells in NSG mice. These data suggest that HuG6.3 may provide a new precision medicine to selectively kill IGHV1-69-encoding G6-id(+) B-CLL cells.

IGHV1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with IGHV utilization shifts and varies by ethnicity.

IGHV polymorphism provides a rich source of humoral immune system diversity. One important example is the IGHV1-69 germline gene where the biased use of alleles that encode the critical CDR-H2 Phe54 (F-alleles) to make broadly neutralizing antibodies (HV1-69-sBnAb) to the influenza A hemagglutinin stem domain has been clearly established. However, whether IGHV1-69 polymorphism can also modulate B cell function and Ab repertoire expression through promoter and copy number (CN) variations has not been reported, nor has whether IGHV1-69 allelic distribution is impacted by ethnicity. Here we studied a cohort of NIH H5N1 vaccinees and demonstrate for the first time the influence of IGHV1-69 polymorphism on V-segment usage, somatic hypermutation and B cell expansion that elucidates the dominance of F-alleles in HV1-69-sBnAbs. We provide evidence that Phe54/Leu54 (F/L) polymorphism correlates with shifted repertoire usage of other IGHV germline genes. In addition, we analyzed ethnically diverse individuals within the 1000 genomes project and discovered marked variations in F- and L- genotypes and CN among the various ethnic groups that may impact HV1-69-sBnAb responses. These results have immediate implications for understanding HV1-69-sBnAb responses at the individual and population level and for the design and implementation of "universal" influenza vaccine.

Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution.

The newly emerging Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes a Severe Acute Respiratory Syndrome-like disease with ∼43% mortality. Given the recent detection of virus in dromedary camels, zoonotic transfer of MERS-CoV to humans is suspected. In addition, little is known about the role of human neutralizing Ab (nAb) pressure as a driving force in MERS-CoV adaptive evolution. Here, we used a well-characterized nonimmune human Ab-phage library and a panning strategy with proteoliposomes and cells to identify seven human nAbs against the receptor-binding domain (RBD) of the MERS-CoV Spike protein. These nAbs bind to three different epitopes in the RBD and human dipeptidyl peptidase 4 (hDPP4) interface with subnanomolar/nanomolar binding affinities and block the binding of MERS-CoV Spike protein with its hDPP4 receptor. Escape mutant assays identified five amino acid residues that are critical for neutralization escape. Despite the close proximity of the three epitopes on the RBD interface, escape from one epitope did not have a major impact on neutralization with Abs directed to a different epitope. Importantly, the majority of escape mutations had negative impacts on hDPP4 receptor binding and viral fitness. To our knowledge, these results provide the first report on human nAbs against MERS-CoV that may contribute to MERS-CoV clearance and evolution. Moreover, in the absence of a licensed vaccine or antiviral for MERS, this panel of nAbs offers the possibility of developing human mAb-based immunotherapy, especially for health-care workers.

Fabrication principles and their contribution to the superior in vivo therapeutic efficacy of nano-liposomes remote loaded with glucocorticoids.

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t(1/2) ~1 h), or a slow, zero-order release rate (t(1/2) ~ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

Amphipathic weak acid glucocorticoid prodrugs remote-loaded into sterically stabilized nanoliposomes evaluated in arthritic rats and in a Beagle dog: a novel approach to treating autoimmune arthritis.

OBJECTIVE: The use of glucocorticoids (GCs) in rheumatoid arthritis is limited by side effects related to unfavorable pharmacokinetics and biodistribution. Liposomal GC formulations have been studied since the 1970s in an attempt to overcome this obstacle, but none has entered clinical use. We undertook this study to determine whether a novel approach could overcome the limitations that have thus far prevented the clinical use of these formulations: low drug:lipid ratio, low encapsulation efficiency, and lack of controlled release. METHODS: We used approximately 80-nm sterically stabilized (pegylated) nanoliposomes (NSSLs), which were remote-loaded with an amphipathic weak acid GC (such as methyl prednisolone hemisuccinate) utilizing an intraliposome (aqueous compartment)-high/extraliposome (bulk medium)-low transmembrane calcium acetate gradient. This unique method actually "traps" the GC in the liposomal aqueous phase as a calcium-GC precipitate. RESULTS: Our liposome formulation exhibited high encapsulation efficiency (94%) and a high drug:lipid mole ratio (0.41) and demonstrated controlled release of the encapsulated GC during systemic circulation and in inflamed paws in rats with adjuvant-induced arthritis. In addition, both in arthritic rats and in a Beagle dog, we showed the pharmacokinetic advantage of using liposomes as GC carriers. Finally, we demonstrated the superior therapeutic efficacy of our liposome formulation over that of free GCs in arthritic rats, both in early and in peak disease stages. CONCLUSION: Amphipathic weak acid GCs remote-loaded into approximately 80-nm NSSLs overcome past limitations of liposomal GC formulations. The unique loading method, which also leads to controlled release, improves the therapeutic effect both systemically and locally. Such a development has great potential for improving GC therapy.

Controlling liposomal drug release with low frequency ultrasound: mechanism and feasibility.

The ability of low-frequency ultrasound (LFUS) to release encapsulated drugs from sterically stabilized liposomes in a controlled manner was demonstrated. Three liposomal formulations having identical lipid bilayer compositions and a similar size ( approximately 100 nm) but differing in their encapsulated drugs and methods of drug loading have been tested. Two of the drugs, doxorubicin and methylpredinisolone hemisuccinate, were remote loaded by transmembrane gradients (ammonium sulfate and calcium acetate, respectively). The third drug, cisplatin, was loaded passively into the liposomes. For all three formulations, a short exposure to LFUS (<3 min) released nearly 80% of the drug. The magnitude of drug release was a function of LFUS amplitude and actual exposure time, irrespective of whether irradiation was pulsed or continuous. Furthermore, no change in liposome size distribution or in the chemical properties of the lipids or of the released drugs occurred due to exposure to LFUS. Based on our results, we propose that the mechanism of release is a transient introduction of porelike defects in the liposome membrane, which occurs only during exposure to LFUS, after which the membrane reseals. This explains the observed uptake of the membrane-impermeable fluorophore pyranine from the extraliposomal medium during exposure to LFUS. The implications of these findings for clinical applications of controlled drug release from liposomes are discussed.

pH determination by pyranine: medium-related artifacts and their correction.

Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) is a water-soluble, membrane-impermeable fluorophore having fluorescent excitation and emission spectra that are highly dependent on medium pH. This combination makes it one of the most commonly used pH-sensitive fluorescent probes to monitor pH and pH changes in biochemical and biophysical research. The pK(a) of this probe is reported to be approximately 7.3, but several studies (including the current one) have shown that this value varies with medium composition. If this is not taken into account, pH determinations based on pyranine may be misleading. We found that in the presence of salts, pK(a) is shifted downward to lower values; therefore, the calculated pH is shifted upward relative to the actual pH as determined by a pH meter. This shift is a consequence of both the type and the concentration of anions and cations that form the salt. Divalent cations cause a larger upward shift in the calculated pH than do monovalent cations. Of all the salts tested, ammonium sulfate has the least effect, and calcium perchlorate has the largest effect, on the pH value calculated by pyranine. Salts are not the only species that affect the pK(a) of pyranine. The presence of the polymer polyethylene glycol (PEG) induces an effect opposite to that of salt (i.e., an upward pK(a) shift), which is expressed as pH being more acidic than that measured by a pH meter. Another nonelectrolyte, dextrose, has no such effect. The effect of both cations and anions can be explained based on their order in the Hofmeister series, whereas the effect of PEG is explained by its high water-binding capacity. Both the ions and PEG change the structure of water and its interaction with pyranine, thereby changing pyranine's apparent pK(a).