Immunological memory diversity in the human upper airway.

The upper airway is an important site of infection, but immune memory in the human upper airway is poorly understood, with implications for COVID-19 and many other human diseases1-4. Here we demonstrate that nasal and nasopharyngeal swabs can be used to obtain insights into these challenging problems, and define distinct immune cell populations, including antigen-specific memory B cells and T cells, in two adjacent anatomical sites in the upper airway. Upper airway immune cell populations seemed stable over time in healthy adults undergoing monthly swabs for more than 1 year, and prominent tissue resident memory T (TRM) cell and B (BRM) cell populations were defined. Unexpectedly, germinal centre cells were identified consistently in many nasopharyngeal swabs. In subjects with SARS-CoV-2 breakthrough infections, local virus-specific BRM cells, plasma cells and germinal centre B cells were identified, with evidence of local priming and an enrichment of IgA+ memory B cells in upper airway compartments compared with blood. Local plasma cell populations were identified with transcriptional profiles of longevity. Local virus-specific memory CD4+ TRM cells and CD8+ TRM cells were identified, with diverse additional virus-specific T cells. Age-dependent upper airway immunological shifts were observed. These findings provide new understanding of immune memory at a principal mucosal barrier tissue in humans.

Characterization of N-glycosylation and amino acid sequence features of immunoglobulins from swine.

The primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to ß-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immunogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features.

Multiplexed azido-group isotopic capture (MAGIC) beads: Selective analysis of azido compounds using a propargyl-based cleavable linker, a proof of concept.

RATIONALE: A cleavable linker is designed and synthesized for the selective capture of azide-containing compounds. This article presents a proof of concept methodology involving the use of peptide-functionalized aminopropyl silica, on which the peptide is constructed by solid-phase peptide synthesis. METHODS: The peptide linker has L-propargylglycine (Pra) at one terminal end to allow the conjugation of azide-containing molecules by copper assisted azide alkyne cycloaddition, also known as click reaction. L-Arginine (Arg) is placed just before Pra to permit the release of the captured product by tryptic cleavage. Three glycine (Gly) residues, as part of the linker, are appended to the silica bead to present a spacer section that allows efficient tryptic cleavage devoid of steric hindrance imposed by the bulky bead. The bead composition is Si-O-propyl-NH-Gly-Gly-Gly-Arg-Pra. RESULTS: This solid-phase material can be used to capture and release azide-functionalized compounds. The beads are first tested on three azido compounds, 2-azido-2-deoxyglucose (ADG), BOC-p-azido-Phe-OH (BAzPhe), where BOC = tert-butoxycarbonyl, and tetraacetylated-N-azidomannosamine (Ac4 ManNAz). Copper-mediated click reaction conditions are used and released products are characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS (MS/MS). CONCLUSIONS: This method allows easy identification of captured compounds based on mass and fragmentation analysis. Moreover, it is useful for the analysis of small azide-containing compounds by MALDI-TOF-MS which may not be possible otherwise due to matrix interferences. The insertion of isotopically labeled Arg residues provides the possibility of multiplex analysis, from which the beads have been called MAGIC (for Multiplexed Azido-Group Isotopic Capture). Copyright © 2016 John Wiley & Sons, Ltd.

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids.

Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble ß-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 µM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.

Dose-dependent regulation of immune memory responses against HIV by saponin monophosphoryl lipid A nanoparticle adjuvant.

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play an important role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (T Mem and B Mem ) and long-lived plasma cells in non-human primates. Robust B Mem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L + OX40 + CD4 T Mem (11-fold), IFNγ + CD4 T Mem (15-fold), IL21 + CD4 T Mem (9-fold), circulating T FH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in non-human primates, which are relevant for human use and next-generation vaccines.

Early antiviral CD4 and CD8 T cell responses are associated with upper respiratory tract clearance of SARS-CoV-2.

T cells are involved in protective immunity against numerous viral infections. Limited data have been available regarding roles of human T cell responses controlling SARS-CoV-2 viral clearance in primary COVID-19. Here, we examined longitudinal SARS-CoV-2 upper respiratory tract viral RNA levels and early adaptive immune responses from 95 unvaccinated individuals with acute COVID-19. Acute SARS-CoV-2-specific CD4 and CD8 T cell responses were evaluated in addition to antibody responses. Most individuals with acute COVID-19 developed rapid SARS-CoV-2-specific T cell responses during infection, and both early CD4 T cell and CD8 T cell responses correlated with reduced upper respiratory tract SARS-CoV-2 viral RNA, independent of neutralizing antibody titers. Overall, our findings indicate a distinct protective role for SARS-CoV-2-specific T cells during acute COVID-19.

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates.

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.