Influence of AAV vector tropism on long-term expression and Fc-γ receptor binding of an antibody targeting SARS-CoV-2.

Long-acting passive immunization strategies are needed to protect immunosuppressed vulnerable groups from infectious diseases. To further explore this concept for COVID-19, we constructed Adeno-associated viral (AAV) vectors encoding the human variable regions of the SARS-CoV-2 neutralizing antibody, TRES6, fused to murine constant regions. An optimized vector construct was packaged in hepatotropic (AAV8) or myotropic (AAVMYO) AAV capsids and injected intravenously into syngeneic TRIANNI-mice. The highest TRES6 serum concentrations (511 µg/ml) were detected 24 weeks after injection of the myotropic vector particles and mean TRES6 serum concentrations remained above 100 µg/ml for at least one year. Anti-drug antibodies or TRES6-specific T cells were not detectable. After injection of the AAV8 particles, vector mRNA was detected in the liver, while the AAVMYO particles led to high vector mRNA levels in the heart and skeletal muscle. The analysis of the Fc-glycosylation pattern of the TRES6 serum antibodies revealed critical differences between the capsids that coincided with different binding activities to murine Fc-γ-receptors. Concomitantly, the vector-based immune prophylaxis led to protection against SARS-CoV-2 infection in K18-hACE2 mice. High and long-lasting expression levels, absence of anti-drug antibodies and favourable Fc-γ-receptor binding activities warrant further exploration of myotropic AAV vector-based delivery of antibodies and other biologicals.

Oriented display of HIV-1 Env trimers by a novel coupling strategy enhances B cell activation and phagocytosis.

Introduction: Conformationally stabilized Env trimers have been developed as antigens for the induction of neutralizing antibodies against HIV-1. However, the non-glycosylated immunodominant base of these soluble antigens may compete with the neutralizing antibody response. This has prompted attempts to couple Env trimers to organic or inorganic nanoparticles with the base facing towards the carrier. Such a site-directed coupling could not only occlude the base of the trimer, but also enhance B cell activation by repetitive display. Methods: To explore the effect of an ordered display of HIV-1 Env on microspheres on the activation of Env-specific B cells we used Bind&Bite, a novel covalent coupling approach for conformationally sensitive antigens based on heterodimeric coiled-coil peptides. By engineering a trimeric HIV-1 Env protein with a basic 21-aa peptide (Peptide K) extension at the C-terminus, we were able to covalently biotinylate the antigen in a site-directed fashion using an acidic complementary peptide (Peptide E) bearing a reactive site and a biotin molecule. This allowed us to load our antigen onto streptavidin beads in an oriented manner. Results: Microspheres coated with HIV-1 Env through our Bind&Bite system showed i) enhanced binding by conformational anti-HIV Env broadly neutralizing antibodies (bNAbs), ii) reduced binding activity by antibodies directed towards the base of Env, iii) higher Env-specific B cell activation, and iv) were taken-up more efficiently after opsonization compared to beads presenting HIV-1 Env in an undirected orientation. Discussion: In comparison to site-directed biotinylation via the Avi-tag, Bind&Bite, offers greater flexibility with regard to alternative covalent protein modifications, allowing selective modification of multiple proteins via orthogonal coiled-coil peptide pairs. Thus, the Bind&Bite coupling approach via peptide K and peptide E described in this study offers a valuable tool for nanoparticle vaccine design where surface conjugation of correctly folded antigens is required.

Association of endothelial activation assessed through endothelin-I precursor peptide measurement with mortality in COVID-19 patients: an observational analysis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been linked to thrombotic complications and endothelial dysfunction. We assessed the prognostic implications of endothelial activation through measurement of endothelin-I precursor peptide (proET-1), the stable precursor protein of Endothelin-1, in a well-defined cohort of patients hospitalized with COVID-19. METHODS: We measured proET-1 in 74 consecutively admitted adult patients with confirmed COVID-19 and compared its prognostic accuracy to that of patients with community-acquired pneumonia (n = 876) and viral bronchitis (n = 371) from a previous study by means of logistic regression analysis. The primary endpoint was all-cause 30-day mortality. RESULTS: Overall, median admission proET-1 levels were lower in COVID-19 patients compared to those with pneumonia and exacerbated bronchitis, respectively (57.0 pmol/l vs. 113.0 pmol/l vs. 96.0 pmol/l, p < 0.01). Although COVID-19 non-survivors had 1.5-fold higher admission proET-1 levels compared to survivors (81.8 pmol/l [IQR: 76 to 118] vs. 53.6 [IQR: 37 to 69]), no significant association of proET-1 levels and mortality was found in a regression model adjusted for age, gender, creatinine level, diastolic blood pressure as well as cancer and coronary artery disease (adjusted OR 0.1, 95% CI 0.0009 to 14.7). In patients with pneumonia (adjusted OR 25.4, 95% CI 5.1 to 127.4) and exacerbated bronchitis (adjusted OR 120.1, 95% CI 1.9 to 7499) we found significant associations of proET-1 and mortality. CONCLUSIONS: Compared to other types of pulmonary infection, COVID-19 shows only a mild activation of the endothelium as assessed through measurement of proET-1. Therefore, the high mortality associated with COVID-19 may not be attributed to endothelial dysfunction by the surrogate marker proET-1.

Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA.

The importance of a balanced TH1/TH2 humoral immune response against the HIV-1 envelope protein (Env) for antibody-mediated HIV-1 control is increasingly recognized. However, there is no defined vaccination strategy to raise it. Since immune checkpoints are involved in the induction of adoptive immunity and their inhibitors (monoclonal antibodies) are licensed for cancer therapy, we investigated the effect of checkpoint blockade after HIV-1 genetic vaccination on enhancement and modulation of antiviral antibody responses. By intraperitoneal administration of checkpoint antibodies in mice we observed an induction of anti-drug antibodies which may interfere with immunomodulation by checkpoint inhibitors. Therefore, we blocked immune checkpoints locally by co-electroporation of DNA vaccines encoding the active soluble ectodomains of programmed cell death protein-1 (PD-1) or its ligand (PD-L1), respectively. Plasmid-encoded immune checkpoints did not elicit a detectable antibody response, suggesting no interference with their immunomodulatory effects. Co-electroporation of a HIV-1 DNA vaccine formulation with soluble PD-L1 ectodomain increased HIV-1 Env-specific TH1 CD4 T cell and IgG2a antibody responses. The overall antibody response was hereby shifted towards a more TH1/TH2 balanced subtype pattern. These findings indicate that co-electroporation of soluble checkpoint ectodomains together with DNA-based vaccines has modulatory effects on vaccine-induced immune responses that could improve vaccine efficacies.

Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

Ligand selectivity of a synthetic CXCR4 mimetic peptide.

The chemokine receptor CXCR4 belongs to the family of seven-transmembrane G-protein coupled receptors (GPCRs). It is activated by its natural ligand SDF-1α. In addition, CXCR4, along with CCR5, serve as coreceptors during HIV-1 entry into its target cell. Recently, we introduced a CXCR4 mimetic peptide, termed CX4-M1, which presents the three extracellular loops (ECLs) of the receptor. CX4-M1 was shown to selectively bind to gp120 of X4-tropic, that is, CXCR4 using, HIV-1, as well as to peptides that present the V3-loops of these gp120 proteins. Furthermore, CX4-M1 selectively inhibits infection of cells with X4-tropic HIV-1. We have now adapted the sequence of the ECLs presented by CX4-M1 to the recently published crystal structure of CXCR4. The binding behavior, as well as the effect on HIV-1 infection, of the resulting peptide (CX4-Mc) was very similar to CX4-M1, validating retrospectively the original design of CX4-M1. A peptide presenting the ECLs of CCR5 (CR5-M), on the other hand, did neither bind to gp120 from X4-tropic HIV-1, nor did it inhibit infection of cells with X4-tropic HIV-1. Furthermore, we could show that CX4-M1, as well as CX4-Mc, but not CR5-M, are selectively recognized by anti-CXCR4 antibodies, bind to SDF-1α, and also inhibit SDF-1α signaling, extending the scope of selective functional CXCR4 mimicry through CX4-M1.