Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.Multiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1–12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13–20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC₅₀ of ∼2–4 µg/mL). Access to infected donors who have high serum titers of bnAbs (21, 22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1–4, 6–8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1–4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG⁺ memory B cells of interest by cell sorting (6, 8, 9, 23, 24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called “GFP-293T^BaL cells,” were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10, 25).The favorable antigenic profile of the soluble BG505 SOSIP.664 gp140 trimer opens the possibility of its use for isolating quaternary-specific antibodies by single-cell sorting (26). To this end, we used BG505 SOSIP.664 gp140 to select for memory B cells from a donor from whom we previously had isolated the trimer-specific bnAbs PGT141–145 (3, 21). (For naming of PGT and PGDM bnAbs, please see SI Materials and Methods, Antibody Nomenclature.) We describe the isolation of previously unidentified somatic variants that are highly divergent from PGT145 and display a range of neutralization breadth and potency, with some being broader and more potent than the previously described PGT145 family members. Overall, the results reveal a mosaic of antibody responses against the trimer-apex site of vulnerability that have important implications for immunogen design in general and for the future optimization of BG505 SOSIP.664 and related native-like trimers as vaccine candidates.     

Dental caries status of Dai preschool children in Yunnan Province,China

Background

The Dai people, one of the ethnic minorities in China, have a population of 1,260,000. They have the same origin as one of the main ethnic groups of Laos and Thailand. Most of the Dai live in Yunnan province, which is located in the less-developed southwestern part of China. This study aimed to describe the oral health status of Dai preschool children in China and the factors that influence their oral health status.

Methods

An oral health survey was performed between 2011 and 2012 to select Dai five-year-old children using multi-stage stratified sampling in Yunnan. Their dental caries experience was measured using the “dmft” index, and severe caries was assessed using the “pa” index, which is modified from the “pufa” index. Oral hygiene status was assessed using the visual plaque index (VPI). A questionnaire to study the children’s socio-demographic background and oral health-related behaviours was completed by the children’s parents.

Results

A total of 833 children were examined. Their caries prevalence was 89% and 49% of the children had carious tooth with pulp involvement. The mean (SD) dmft score was 7.0 (5.3). Higher dmft scores were found among children who were girls, were currently bottle-fed, took daily sweet snacks, had higher VPI scores, and had visited a dentist within the last year.

Conclusions

The caries prevalence and experience of the five-year-old Dai children in Yunnan, China was high, and almost half had severe caries. The caries experience was associated with gender, snack habits, dental visit habits, and oral hygiene status.

     

A Decrease in the Percentage of CD3+ Cells Is Correlated With Clinical Improvement During Plasmapheresis in Patients With Myasthenia Gravis

Plasmapheresis not only removes circulating antibodies but also modulates cellular immunity, including lymphocyte subsets. To investigate the effect of double‐filtration plasmapheresis (DFPP) on the ratio of lymphocyte subsets in patients with myasthenia gravis (MG), we examined the percentages of B‐cells, T‐cells, T helper (Th) cells, T suppressor (Ts) cells, natural killer (NK) cells, NKT cells, and Th/Ts ratio before and after a single DFPP session and after a course of DFPP. A total of 26 patients were recruited; their peripheral blood lymphocyte subsets were assayed using flow cytometry. After a single session of DFPP treatment, the percentages of T‐cells (P = 0.0200), Th cells (P = 0.0178), and the Th/Ts ratio (P = 0.0309) decreased significantly, whereas the percentage of NK cells (P = 0.0007) increased significantly. More importantly, after one course of DFPP treatment, the reduced clinical quantitative MG (QMG) score was correlated with the decrease of the percentage of T‐cells (r = 0.5005, P = 0.0092). Fourteen thymectomized MG patients had decreased percentages of T‐cells (P = 0.0304) and Th cells (P = 0.0444), whereas they had increased NK cells (P = 0.0197) after a single DFPP session. Here, transiently decreased percentages of T‐cells after the full DFPP course could enhance the effectiveness of plasmapheresis for MG patients.     

Recent Trend of Necrotizing Fasciitis in Taiwan: Focus on Monomicrobial Klebsiella pneumoniae Necrotizing Fasciitis

Background.?Necrotizing fasciitis (NF) is a rapidly progressive, life-threatening soft-tissue infection that is traditionally caused by group A Streptococcus (GAS) or mixed aerobic/anaerobic bacteria. Monomicrobial Klebsiella pneumoniae NF (KP-NF) has been reported since 1996 but has not yet been systematically studied. Methods.?We retrospectively studied consecutive NF cases treated at a university hospital in Taiwan during 1997-2010 and investigated the clinical characteristics and outcomes associated with monomicrobial KP-NF, using monomicrobial GAS-NF as a reference. We also analyzed the virulence gene profiles of the isolated K. pneumoniae strains. Results.?Of 134 NF cases, 88 were monomicrobial, of which the most common pathogens were GAS (n?=?16) and K. pneumoniae (n?=?15). Monomicrobial KP-NF entailed a moderate risk of limb loss (20% vs 25%; P?=?1.000) and high mortality (47% vs 19%; P?=?.135), and it was more likely to involve bacteremia (80% vs 31%; P?=?.011), concomitant distant abscesses (27% vs 0%; P?=?.043), and underlying immunocompromising conditions (100% vs 63%; P?=?.018), compared with GAS-NF. The isolated K. pneumoniae strains (n?=?10) were of capsular polysaccharides genotype K1 (n?=?4), K54/K20/K5 (n?=?4), K2 (n?=?1), and K16 (n?=?1). All strains carried rmpA, iucABCDiutA, and iroA. Genotype K1 strains had a significantly higher risk of concomitant distant abscesses, compared with non-K1 strains (75% vs 0%; P?=?.033). Conclusions.?K. pneumoniae has become a common pathogen of monomicrobial NF in Taiwan. Physicians treating patients with monomicrobial KP-NF should be aware of the risk of concomitant distant abscesses, particularly in cases caused by genotype K1.