Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants.

BACKGROUND: HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. RESULTS: N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. CONCLUSIONS: The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries.

Feasibility and acceptability of a pilot, peer-led HIV self-testing intervention in a hyperendemic fishing community in rural Uganda.

BACKGROUND: Novel interventions are needed to reach young people and adult men with HIV services given the low HIV testing rates in these population sub-groups. We assessed the feasibility and acceptability of a peer-led oral HIV self-testing (HIVST) intervention in Kasensero, a hyperendemic fishing community (HIV prevalence: 37-41%) in Rakai, Uganda. METHODS: This study was conducted among young people (15-24 years) and adult men (25+ years) between May and August 2019. The study entailed distribution of HIVST kits by trained "peer-leaders," who were selected from existing social networks and trained in HIVST distribution processes. Peer-leaders received up to 10 kits to distribute to eligible social network members (i.e. aged 15-24 years if young people or 25+ years if adult man, not tested in the past 3 months, and HIV-negative or of unknown HIV status at enrolment). The intervention was evaluated against the feasibility benchmark of 70% of peer-leaders distributing up to 70% of the kits that they received; and the acceptability benchmark of >80% of the respondents self-testing for HIV. RESULTS: Of 298 enrolled into the study at baseline, 56.4% (n = 168) were young people (15-24 years) and 43.6% (n = 130) were adult males (25+ years). Peer-leaders received 298 kits and distributed 296 (99.3%) kits to their social network members. Of the 282 interviewed at follow-up, 98.2% (n = 277) reported that they used the HIVST kits. HIV prevalence was 7.4% (n = 21). Of the 57.1% (n = 12) first-time HIV-positives, 100% sought confirmatory HIV testing and nine of the ten (90%) respondents who were confirmed as HIV-positive were linked to HIV care within 1 week of HIV diagnosis. CONCLUSION: Our findings show that a social network-based, peer-led HIVST intervention in a hyperendemic fishing community is highly feasible and acceptable, and achieves high linkage to HIV care among newly diagnosed HIV-positive individuals.

An innovative vending machine-based HIV testing and intervention service in China: anonymous urine collection kits distributed at universities.

To find more effective test and intervention measures, and to achieve the first 90 of the 90-90-90 target, this study was conducted for the first time to develop and assess an innovative HIV anonymous urine test service-based vending machine and Internet at universities of China. From June to December 2016, 11 vending machines were placed in 7 pilot universities in Beijing, Sichuan, Yunnan and Heilongjiang provinces. A total of 957 HIV urine collection kits were dispensed free and also through vending machines and 378 (39.5%) urine samples were returned and 376 (99.5%) of them were qualified to be tested for HIV antibody in professional laboratories. Participants searched for confidential test results using an ID code online. Only seven (1.86%) urine samples were positive. Monitoring data showed 67.8% (255/376) participants searched for test results online, 72.2% of kits were purchased in dormitory buildings and 27.8% were purchased in teaching buildings and 88.9% were purchased between 21:00 and 24:00. In conclusion, this study analyzes the acceptability, feasibility and effectiveness of HIV testing and intervention service.

HIV screening among patients seeking care at Xuanwu Hospital: A cross-sectional study in Beijing, China, 2011-2016.

OBJECTIVES: One-third of people living with HIV in China are still unaware of their status, so we sought to better understand HIV testing in the general hospital setting in China. METHODS: A cross-sectional study was conducted using the electronic medical records of all patients who attended Xuanwu Hospital in Beijing, January 1, 2011 to December 31, 2016. HIV screening and detection rates and characteristics of patients diagnosed with HIV were assessed. RESULTS: Overall, 235,961 patients were screened, for a screening rate of 1.4%. Although most were outpatients (98.4%), screening rate was higher among inpatients (70.0% versus 0.4%), and highest in internal medicine (36.1%) and surgery (33.3%) departments. A total of 140 patients were diagnosed with HIV, for a detection rate of 5.93 per 10,000. Detection rates were highest among outpatients (9.34 per 10,000), and patients attending the dermatology and sexually transmitted infection (STI) department (153.85 per 10,000). Most diagnoses were made among males (91.4%), aged 20-39 (67.1%), who reported becoming infected through homosexual contact (70.0%). CONCLUSIONS: HIV screening in China's general hospitals needs to be improved. More focus should be placed on screening outpatients, especially in the dermatology and STI department, and young men.

Identification of a HIV Gp41-Specific Human Monoclonal Antibody With Potent Antibody-Dependent Cellular Cytotoxicity.

Antibody-Dependent Cellular Cytotoxicity (ADCC) is a major mechanism of protection against viral infections in vivo. Identification of HIV-1-specific monoclonal antibodies (mAbs) with potent ADCC activity may help develop an effective HIV-1 vaccine. In present study, we isolated such human mAb, designated E10, from an HIV-1-infected patient sample by single B cell sorting and single cell PCR. E10 bound to gp140 trimer and linear peptides derived from gp41 membrane proximal external region (MPER). E10 epitope (QEKNEQELLEL) overlapped with mAb 2F5 epitope. However, E10 differentiated from 2F5 in neutralization breadth and potency, as well as ADCC activity. E10 showed low neutralization activity and narrow spectrum of neutralization compared to 2F5, but it mediated higher ADCC activity than 2F5 at low antibody concentration. Fine mapping of E10 epitope may potentiate MPER-based subunit vaccine development.

V2-Directed Vaccine-like Antibodies from HIV-1 Infection Identify an Additional K169-Binding Light Chain Motif with Broad ADCC Activity.

Antibodies that bind residue K169 in the V2 region of the HIV-1 envelope correlated with reduced risk of infection in the RV144 vaccine trial but were restricted to two ED-motif-encoding light chain genes. Here, we identify an HIV-infected donor with high-titer V2 peptide-binding antibodies and isolate two antibody lineages (CAP228-16H/19F and CAP228-3D) that mediate potent antibody-dependent cell-mediated cytotoxicity (ADCC). Both lineages use the IGHV5-51 heavy chain germline gene, similar to the RV144 antibody CH58, but one lineage (CAP228-16H/19F) uses a light chain without the ED motif. A cocrystal structure of CAP228-16H bound to a V2 peptide identified a IGLV3-21 gene-encoded DDxD motif that is used to bind K169, with a mechanism that allows CAP228-16H to recognize more globally relevant V2 immunotypes. Overall, these data further our understanding of the development of cross-reactive, V2-binding, antiviral antibodies and effectively expand the human light chain repertoire able to respond to RV144-like immunogens.

[Analysis on influencing factors that leading to nonspecific responses to indeterminate results of HIV antibodies].

Objective: To identify the influencing factors that leading to nonspecific responses to indeterminate HIV antibody tests, to provide scientific evidence for the differential diagnosis of HIV infection and control strategy. Methods: A case control study was conducted. The samples of HIV antibody indeterminate in confirmed Western blot (WB) tests, but were negative in HIV nucleic acid tests, were collected as HIV antibody indeterminate group from WB results of HIV confirmatory laboratories of Fujian province in 2015-2016. The general population matched group with HIV antibody screening negative samples and WB negative matched group with WB negative samples were selected as the two compared groups by matching gender and age from HIV antibody screening in Fujian province in the same period. Blood concentrations of hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (HCV) antibody, anti-treponema pallidum (TP) antibody, antinuclear antibody (ANA), anti-human T-cell leukemia virus (HTLV) antibody, and alpha-fetoprotein (AFP) were detected by using enzyme-linked immunosorbent assay (ELISA). χ(2) test and multivariate non-conditional logistic regression analysis were performed to identify the influencing factors that leading to nonspecific responses, to indeterminate HIV antibody tests. Results: A total of 13 WB band patterns were observed in 110 HIV antibody indeterminate samples, in which a single p24 band (58.18%, 64/110), a single gp160 band (17.27%, 19/110) and a single p17 band (7.27%, 8/110) were the three most common patterns. The positive rate of anti-TP antibody was significantly higher in HIV antibody indeterminate samples than general population control group and WB negative control group (10.91%, 12/110 vs. 1.77%, 4/226 and 3.64%, 4/110), compared with two control groups (χ(2)=13.627 and 4.314, P<0.05). The positive rate of AFP was significantly higher in HIV antibody indeterminate samples than general population control group (18.18%, 20/110 vs. 0.44%, 1/226, χ(2)=39.736, P<0.05), the different was not significant compared with WB negative control group (18.18%, 20/110 vs. 23.64%, 26/110, χ(2)=0.990, P>0.05) While no significant differences were found between HIV antibody indeterminate group and two control groups in terms of the positive rates of ANA, HBsAg, anti-HCV antibody or anti-HTLV antibody. Conclusions: The influencing factors that leading to nonspecific responses to indeterminate HIV antibody tests appeared complicate, and the anti-TP antibody positivity might be an influencing factor responsible for nonspecific responses to indeterminate HIV antibody tests.

Evaluation of the vitros hiv combo 4th generation test for the identification of HIV infections.

BACKGROUND: Simultaneous detection of HIV 1 and 2 antibodies and HIV-1 p-24 antigen in the 4th generation tests is particularly effective for the identification of early acute HIV infections while maintaining accurate detection of long-established infections. OBJECTIVES: The aim of this study was to evaluate the new 4th generation VITROS HIV Combo test from Ortho-Clinical Diagnostics by comparing its results with those obtained using a 3rd generation HIV 1/2 antibody test (VITROS Anti HIV 1 + 2 from Ortho-Clinical Diagnostics) and a 4th generation test (LIAISON XL HIV Ab/Ag, DiaSorin) currently used in the Microbiology Unit of Legnano Hospital. STUDY DESIGN: One thousand and three samples of the normal daily routine (Group 1) were analyzed simultaneously with the three systems. The concordant and discordant sample results were further tested using Western blot and HIV-RNA assay (Roche). One hundred samples (Group 2) of known HIV positive subjects (63 of subtype B, 37 subtype non-B, and 51 with positive viraemia) and 50 samples (Group 3) with indeterminate Western blot were also examined using the three systems. From Group 3, 24 samples were collected from patients diagnosed with acute infection. RESULTS: The overall agreement between the three systems was 99.4% (99.5% in group 1, 100% in group 2 and 96.6% in group 3) with a coefficient Fleiss Kappa of 0.9814. Notably, the VITROS HIV Combo test was positive in all known HIV positive samples of group 2 without any statistically significant difference in the values of the sample/cut off ratios between the B and non-B subtypes and between the positive and negative viraemia samples in established infections. The VITROS HIV Combo test was also positive in all samples of patients with acute infection in group 3. CONCLUSIONS: The VITROS HIV Combo test has shown comparable performance to the other two assays in use of 3rd and 4th generation tests and is able to correctly identify both acute and established HIV infections independently of viraemia and HIV subtype.

Dimeric Fcγ Receptor Enzyme-Linked Immunosorbent Assay To Study HIV-Specific Antibodies: A New Look into Breadth of Fcγ Receptor Antibodies Induced by the RV144 Vaccine Trial.

Ab-dependent cellular cytotoxicity (ADCC) responses are of growing interest in the HIV vaccine field but current cell-based assays are usually difficult to reproduce across laboratories. We developed an ELISA and multiplex assay to model the cross-linking of Fcγ receptors (FcγR) by Abs, which is required to initiate an ADCC response. Our FcγR dimer ELISA readily detected Abs in samples from two separate cohorts of the partially efficacious Thai RV144 HIV vaccine efficacy trial. The FcγR dimer-binding Abs induced by the RV144 regimen correlated well with a functional measure of ADCC as well as IgG subclasses. The high-throughput multiplex assay allowed us to simultaneously measure FcγR dimer-binding Abs to 32 different HIV Ags, providing a measure of the breadth of FcγR-binding Abs induced by the RV144 trial. FcγR-binding Abs specific to V regions 1 and 2 were strongly associated with increased breadth of recognition of different Env proteins, suggesting anti-V regions 1 and 2 Abs may be a marker of ADCC breadth. This FcγR dimer provides an important tool for the further analysis and refinement of ADCC-inducing HIV and other antiviral vaccine regimens.

Performance evaluation of the point-of-care INSTI™ HIV-1/2 antibody test in early and established HIV infections.

BACKGROUND: The flow-through INSTI™ HIV-1/HIV-2 Rapid Antibody (INSTI) test is a 60s FDA-approved test for HIV-1 and HIV-2 antibody testing using whole blood and plasma. OBJECTIVE: We evaluated the performance of INSTI using plasma and simulated whole blood specimens. STUDY DESIGN: INSTI's performance in plasma specimens from commercial seroconversion panels was assessed by estimating the relative sensitivity using a 50% cumulative frequency analysis and by comparing its performance with other FDA-approved rapid tests (RTs). INSTI was further evaluated using 320 HIV-1 plasma specimens collected during a cross-sectional study and with 107 HIV-1 and 24 HIV-2 simulated whole blood specimens. Sensitivity and specificity were calculated using 615 known HIV-1 group M/O and 80 HIV-2 (Western blot (WB)-positive), and 497 HIV-negative plasma specimens, respectively. RESULTS: In HIV-1 seroconversion panels, INSTI became reactive 9days before a positive WB. When compared to FDA-approved antibody-based lateral flow RTs, INSTI detected significantly more early infections. Among HIV-1-infected cross-sectional plasma samples, INSTI detected 23 (27%) of 85 Architect-positive/Multispot-negative or indeterminate specimens. For plasma specimens, the sensitivity was 99.84% for HIV-1 and 100% for HIV-2, and the specificity was 99.80%. Using simulated whole blood from seroconverters, INSTI performed similarly to plasma. CONCLUSIONS: INSTI performed significantly better than antibody-based lateral flow RTs during early stages of seroconversion. Sensitivity and specificity were within the manufacturer's reported ranges. Considering the observed test performance and the almost immediate results, INSTI is an accurate option to detect HIV-1/HIV-2 antibodies in point-of-care settings where lab testing is not feasible.

Real-world performance of the new US HIV testing algorithm in medical settings.

BACKGROUND: Our medical center laboratory recently adapted its 24/7, two-hourly testing program to use an ARCHITECT-Multispot-viral load (AR-MS-VL) algorithm in place of a previous rapid test-immunofluorescence (RT-IF) algorithm. OBJECTIVES: We evaluated screening test performance, acute case detection, turnaround time and ability to resolve HIV status under the new algorithm. STUDY DESIGN: We considered consecutive HIV tests from January to November 2015. AR-MS-VL results at Zuckerberg San Francisco General Hospital and Trauma Center (ZSFG) were compared with RT-IF results at ZSFG and also with AR-MS-VL results in the recently completed CDC Screening Targeted Populations to Interrupt On-going Chains of HIV Transmission with Enhanced Partner Notification (STOP) Study for targeted testing of MSM at publicly funded testing sites in San Francisco. RESULTS: Among 21,985 HIV tests performed at ZSFG, 16,467 were tested by RT-IF and 5518 by AR-MS-VL. There were 321 HIV infections detected, of which 274 (84%) were known HIV+ cases, and 47 were newly identified HIV infections. Considering only patients of HIV-negative or -unknown status, prevalence was 0.22%. Under the AR-MS-VL algorithm, turnaround times for screening results and full algorithm results were 3 and 21h; status-unresolved cases were reduced (from 47% to 22%) compared with the RT-IF algorithm. The positive predictive value (PPV) of a new-positive AR screening test was low (0.44) at ZSFG, where no acute infections were detected. At STOP Study sites where HIV prevalence was higher and acute infection was more common, the AR PPV was higher (0.93). All 24 false-positive AR screening tests at ZSFG had a signal/cutoff (S/CO) ratio of <15 and all 88 true-positive tests had S/CO ratio >15. Of 62 acute infections in the STOP Study, 23 (37%) had an S/CO<15. DISCUSSION: An AR-MS-VL algorithm is feasible and can return rapid results in a large medical center. In this setting, reactive 4th generation assay tests that are negative for HIV antibodies are typically false-positive with low S/CO ratios.

[HIV antibody detection results in patients seeking medical care in Peking Union Medical College Hospital, 2003-2014].

Objective: To better understand the infection status of HIV in the patients seeking medical care in Peking Union Medical College Hospital. Methods: The HIV detection data of the patients in the hospital from 2003-2014 were collected for a statistical analysis with software SPSS 19.0. Results: A total of 715 421 patients were screened, and 1 012 (0.14%) patients were HIV positive, and HIV infection were confirmed in 776 (0.11%) patients by Western Blot testing. The detection rate of HIV infection increased from 0.05% in 2003 to 0.17% in 2014 (trend χ(2)=66.83 , P=0.000), and the increase during 2012-2014 was obvious. Of the 776 newly diagnosed HIV-infected individuals, 631 (81.31%) were men and 145 (18.69%) were women. The percentage of the males infected with HIV increased from 50.00% to 90.26% (trend χ(2)=58.41, P=0.000). The median age was 36 years (interquartile range: 27-43), and the age group 18-50 years were mostly affected. In the 776 patients infected with HIV, 634 (81.70% ) were infected through sexual contacts, and the proportion of sexual transmissions increased with year (trend χ(2)=126.38, P=0.000). The proportion of infected men who have sex with men (MSM) increased from 0% in 2003 to 53.90% in 2014 (trend χ(2)=11.96, P=0.001), similar to the trend in western countries. The proportion of infected patients who were not married increased from 18.75% to 42.21% (trend χ(2)=43.74, P=0.000). The top three source departments of HIV/AIDS cases were internal medicine (51.03%), emergency room (18.30%) and dermatology (13.53%). The proportion of the HIV/AIDS patients from department of gynecology and obstetrics declined from 18.75% in 2003 to 2.60% in 2014. No HIV/AIDS patients were detected in department of surgery, department of otorhinolaryngology, department of ophthalmology, department of stomatology and health examination center in 2003, but 14 cases (9.10%), 11 cases (7.14%) and 4 cases (2.60%) were detected in these departments respectively in 2014. Conclusion: The HIV detection rate increased with year in Peking Union Medical College Hospital, suggesting the necessity of strengthened HIV test in general hospitals. MSM are the population at high risk, to whom more attention should be paid.

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape.

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design.

Targeted Isolation of Antibodies Directed against Major Sites of SIV Env Vulnerability.

The simian immunodeficiency virus (SIV) challenge model of lentiviral infection is often used as a model to human immunodeficiency virus type 1 (HIV-1) for studying vaccine mediated and immune correlates of protection. However, knowledge of the structure of the SIV envelope (Env) glycoprotein is limited, as is knowledge of binding specificity, function and potential efficacy of SIV antibody responses. In this study we describe the use of a competitive probe binding sort strategy as well as scaffolded probes for targeted isolation of SIV Env-specific monoclonal antibodies (mAbs). We isolated nearly 70 SIV-specific mAbs directed against major sites of SIV Env vulnerability analogous to broadly neutralizing antibody (bnAb) targets of HIV-1, namely, the CD4 binding site (CD4bs), CD4-induced (CD4i)-site, peptide epitopes in variable loops 1, 2 and 3 (V1, V2, V3) and potentially glycan targets of SIV Env. The range of SIV mAbs isolated includes those exhibiting varying degrees of neutralization breadth and potency as well as others that demonstrated binding but not neutralization. Several SIV mAbs displayed broad and potent neutralization of a diverse panel of 20 SIV viral isolates with some also neutralizing HIV-2(7312A). This extensive panel of SIV mAbs will facilitate more effective use of the SIV non-human primate (NHP) model for understanding the variables in development of a HIV vaccine or immunotherapy.

The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Structural analysis of the unmutated ancestor of the HIV-1 envelope V2 region antibody CH58 isolated from an RV144 vaccine efficacy trial vaccinee.

Human monoclonal antibody CH58 isolated from an RV144 vaccinee binds at Lys169 of the HIV-1 Env gp120 V2 region, a site of vaccine-induced immune pressure. CH58 neutralizes HIV-1 CRF_01 AE strain 92TH023 and mediates ADCC against CD4 + T cell targets infected with CRF_01 AE tier 2 virus. CH58 and other antibodies that bind to a gp120 V2 epitope have a second light chain complementarity determining region (LCDR2) bearing a glutamic acid, aspartic acid (ED) motif involved in forming salt bridges with polar, basic side amino acid side chains in V2. In an effort to learn how V2 responses develop, we determined the crystal structures of the CH58-UA antibody unliganded and bound to V2 peptide. The structures showed an LCDR2 structurally pre-conformed from germline to interact with V2 residue Lys169. LCDR3 was subject to conformational selection through the affinity maturation process. Kinetic analyses demonstrate that only a few contacts were responsible for a 2000-fold increase in KD through maturation, and this effect was predominantly due to an improvement in off-rate. This study shows that preconformation and preconfiguration can work in concert to produce antibodies with desired immunogenic properties.

Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

OBJECTIVE: To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. DESIGN: Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. METHODS: Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. RESULTS: Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. CONCLUSIONS: Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.