Phase 1 trial evaluating safety and pharmacokinetics of HIV-1 broadly neutralizing mAbs 10E8VLS and VRC07-523LS.

BACKGROUNDBroadly neutralizing monoclonal antibodies (bNAbs) represent a promising strategy for HIV-1 immunoprophylaxis and treatment. 10E8VLS and VRC07-523LS are bNAbs that target the highly conserved membrane-proximal external region (MPER) and the CD4-binding site of the HIV-1 viral envelope glycoprotein, respectively.METHODSIn this phase 1, open-label trial, we evaluated the safety and pharmacokinetics of 5 mg/kg 10E8VLS administered alone, or concurrently with 5 mg/kg VRC07-523LS, via s.c. injection to healthy non-HIV-infected individuals.RESULTSEight participants received either 10E8VLS alone (n = 6) or 10E8VLS and VRC07-523LS in combination (n = 2). Five (n = 5 of 8, 62.5%) participants who received 10E8VLS experienced moderate local reactogenicity, and 1 participant (n = 1/8, 12.5%) experienced severe local reactogenicity. Further trial enrollment was stopped, and no participant received repeat dosing. All local reactogenicity resolved without sequelae. 10E8VLS retained its neutralizing capacity, and no functional anti-drug antibodies were detected; however, a serum t1/2 of 8.1 days was shorter than expected. Therefore, the trial was voluntarily stopped per sponsor decision (Vaccine Research Center, National Institute of Allergy and Infectious Diseases [NIAID], NIH). Mechanistic studies performed to investigate the underlying reason for the reactogenicity suggest that multiple mechanisms may have contributed, including antibody aggregation and upregulation of local inflammatory markers.CONCLUSION10E8VLS resulted in unexpected reactogenicity and a shorter t1/2 in comparison with previously tested bNAbs. These studies may facilitate identification of nonreactogenic second-generation MPER-targeting bNAbs, which could be an effective strategy for HIV-1 immunoprophylaxis and treatment.TRIAL REGISTRATIONClinicaltrials.gov, accession no. NCT03565315.FUNDINGDivision of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.

Structures of HIV-1 Neutralizing Antibody 10E8 Delineate the Mechanistic Basis of Its Multi-Peak Behavior on Size-Exclusion Chromatography.

Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope.

Immune Monitoring Reveals Fusion Peptide Priming to Imprint Cross-Clade HIV-Neutralizing Responses with a Characteristic Early B Cell Signature.

The HIV fusion peptide (FP) is a promising vaccine target. FP-directed monoclonal antibodies from vaccinated macaques have been identified that neutralize up to ∼60% of HIV strains; these vaccinations, however, have involved ∼1 year with an extended neutralization-eclipse phase without measurable serum neutralization. Here, in 32 macaques, we test seven vaccination regimens, each comprising multiple immunizations of FP-carrier conjugates and HIV envelope (Env) trimers. Comparisons of vaccine regimens reveal FP-carrier conjugates to imprint cross-clade neutralizing responses and a cocktail of FP conjugate and Env trimer to elicit the earliest broad responses. We identify a signature, appearing as early as week 6 and involving the frequency of B cells recognizing both FP and Env trimer, predictive of vaccine-elicited breadth ∼1 year later. Immune monitoring of B cells in response to vaccination can thus enable vaccine insights even in the absence of serum neutralization, here identifying FP imprinting, cocktail approach, and early signature as means to improve FP-directed vaccine responses.

Preclinical Development of a Fusion Peptide Conjugate as an HIV Vaccine Immunogen.

The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) - when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) - to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.

Characterization of AEBSF-antibody modifications for a protease inhibitor supplementation strategy.

Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.

Titer measurement of HIV-1 envelope trimeric glycoprotein in cell culture media by a new tandem ion exchange and size exclusion chromatography (IEC-SEC) method.

An efficient and specific liquid chromatography (LC)-based assay was developed to monitor the production of recombinant HIV-1 trimeric envelope glycoprotein (HIV Env trimer), a candidate vaccine for HIV-1 infection, in cell culture media to support scale-up process development. In this method, titer measurement was achieved by coupling a weak anion exchange chromatography (IEC) column with a size exclusion chromatography (SEC) column. This assay was specific, accurate, precise, and has been qualified for its intended purpose, with a limit of quantification (LOQ) of 10 µg/mL. This tandem separation strategy offered a reliable and timely analytical support to directly monitor the titer of HIV Env trimer during cell growth, without any extra sample purification steps.

Overcoming Challenges in Structural Characterization of HIV-1 Envelope Glycoprotein by LC-MS/MS.

Characterization of HIV Env glycoprotein with 28 glycosylation sites is the essential step of structure-based vaccine design programs. A comprehensive LC-MS/MS peptide mapping analysis was applied to assess the primary sequence, glycosylation profiles, and glycosite occupancy of Env to ensure the adequate mimicking of the native immunogen. Another structural feature was reported, related to its cleaved subunits within the trimeric assembly. We bring attention to the importance of thorough inspection of the results generated by the informatics tools which are currently available for the biopharmaceutical characterization. The complexity of Env translates into a vast amount of data with occasional information gaps that could not possibly be filled by means of the automatic data analysis. A series of data validation steps was applied, followed by the illustrations on how the high-quality results may be misinterpreted. It was shown that the glycan sites can only be characterized to a certain limit, and that any claim of full structural characterization of this molecule beyond these limits should be treated with caution. Following the result verification, the percent glycan occupancy was reported for 25 N-glycan sites, including 3 critical antibody-recognition sites. The exact glycan profiles were provided for 20 individual sites, whereas only the glycosylation type could be deduced for 5 sites, dictated by their location within Env sequence. The distribution of the unprocessed high mannose-type glycans correlated with the expected "mannose patch." Experimental procedure optimization and a workflow for glycan characterization with a focus on stringent data testing are presented in the current study.

Quantification of residual AEBSF-related impurities by reversed-phase liquid chromatography.

During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 µM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.

Development of a New Tandem Ion Exchange and Size Exclusion Chromatography Method To Monitor Vaccine Particle Titer in Cell Culture Media.

A new tandem chromatography method was developed to directly measure the titers of various vaccine candidate molecules in cell culture without a prior purification step. The method utilized a strong anion exchange chromatography (IEC) column in tandem with a size exclusion chromatography (SEC) column to efficiently separate the nanoparticle and virus-like particle (VLP) vaccine molecules from host cell proteins and other components in the cell culture media. The dual (charge and hydrodynamic size) separation mode was deemed necessary to achieve good separation of the vaccine product for quantitation. The method development and quality assessment illustrated herein was focused on the influenza vaccine candidate H1ssF, a hemagglutinin (group 1) stabilized stem molecule fused to ferritin to form nanoparticles. This newly established method was then successfully applied to several vaccine candidate developmental projects, such as the hemagglutinin-ferritin (HAF) nanoparticle and encephalitic alphavirus VLP-based vaccines. This IEC-SEC strategy was established as a platform approach for direct titer measurement of novel vaccine molecules in cell culture.

Environmental tobacco smoke and cardiometabolic risk in young children: results from a survey in south-west Germany.

AIMS: We explored the association between exposure to environmental tobacco smoke (ETS) and various cardiometabolic biomarkers in 10-year-old children. METHODS AND RESULTS: A population-based cross-sectional study was carried out. Data on ETS exposure and potential confounders were collected by parental questionnaire. Adiponectin, leptin, markers of inflammation, apolipoproteins (apo) AI and B, and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) were measured. Linear and logistic regression models were applied using the 90th percentile as a cut-off point except for adiponectin and apoAI (10th percentile). In linear models, ETS exposure was significantly associated with increasing plasma concentrations of leptin, C-reactive protein, fibrinogen, interleukin (IL)-6, and Lp-PLA(2). When compared with none, ETS exposure of more than 10 cigarettes per day was associated with elevated concentrations of leptin (OR 6.40; 95% CI, 2.67-15.39), C-reactive protein (OR 3.17; 95% CI, 1.31-7.68), Lp-PLA(2) (OR 2.97 95% CI, 1.32-6.68), low adiponectin (OR 2.69; 95% CI, 1.10-6.57), and low apoAI (OR 4.48; 95% CI, 2.16-10.85). Increasing dose of ETS exposure was related to an increasing number of abnormal cardiometabolic markers. CONCLUSION: Among children, ETS exposure was associated with a low-grade inflammatory response and altered markers of lipid metabolism, which may initiate atherosclerosis in early life. However, longitudinal studies are necessary to determine the potential causal relevance of these associations.