Intranasal Delivery of MVA Vector Vaccine Induces Effective Pulmonary Immunity Against SARS-CoV-2 in Rodents

Antigen-specific tissue-resident memory T cells (Trms) and neutralizing IgA antibodies provide the most effective protection of the lungs from viral infections. To induce those essential components of lung immunity against SARS-CoV-2, we tested various immunization protocols involving intranasal delivery of a novel Modified Vaccinia virus Ankara (MVA)-SARS-2-spike vaccine candidate. We show that a single intranasal MVA-SARS-CoV-2-S application in mice strongly induced pulmonary spike-specific CD8+ T cells, albeit restricted production of neutralizing antibodies. In prime-boost protocols, intranasal booster vaccine delivery proved to be crucial for a massive expansion of systemic and lung tissue-resident spike-specific CD8+ T cells and the development of Th1 - but not Th2 - CD4+ T cells. Likewise, very high titers of IgG and IgA anti-spike antibodies were present in serum and broncho-alveolar lavages that possessed high virus neutralization capacities to all current SARS-CoV-2 variants of concern. Importantly, the MVA-SARS-2-spike vaccine applied in intramuscular priming and intranasal boosting treatment regimen completely protected hamsters from developing SARS-CoV-2 lung infection and pathology. Together, these results identify intramuscular priming followed by respiratory tract boosting with MVA-SARS-2-S as a promising approach for the induction of local, respiratory as well as systemic immune responses suited to protect from SARS-CoV-2 infections

Manifold Roles of CCR7 and Its Ligands in the Induction and Maintenance of Bronchus-Associated Lymphoid Tissue

Summary: The processes underlying the development and maintenance of tertiary lymphoid organs are incompletely understood. Using a Ccr7 knockout/knockin approach, we show that spontaneous bronchus-associated lymphoid tissue (BALT) formation can be caused by CCR7-mediated migration defects of dendritic cells (DCs) in the lung. Plt/plt mice that lack the CCR7 ligands CCL19 and CCL21-serine do not form BALT spontaneously because lung-expressed CCL21-leucine presumably suffices to maintain steady-state DC egress. However, plt/plt mice are highly susceptible to modified vaccinia virus infection, showing enhanced recruitment of immune cells as well as alterations in CCR7-ligand-mediated lymphocyte egress from the lungs, leading to dramatically enhanced BALT. Furthermore, we identify two independent BALT homing routes for blood-derived lymphocytes. One is HEV mediated and depends on CCR7 and L-selectin, while the second route is via the lung parenchyma and is independent of these molecules. Together, these data provide insights into CCR7/CCR7-ligand-orchestrated aspects in BALT formation. : Fleige et al. demonstrate that CCR7 and its ligands CCL19, CCL21-serine, and CCL21-leucine orchestrate multiple steps during induction and maintenance of bronchus-associated lymphoid tissue (BALT) including DC-based initial developmental processes as well as homing of blood-derived lymphocytes via HEVs to established BALT. Keywords: BALT, CCR7, CCL21, DCs, MVA, plt/plt, HEV

Human genetic adaptation related to cellular zinc homeostasis.

SLC30A9 encodes a ubiquitously zinc transporter (ZnT9) and has been consistently suggested as a candidate for positive selection in humans. However, no direct adaptive molecular phenotype has been demonstrated. Our results provide evidence for directional selection operating in two major complementary haplotypes in Africa and East Asia. These haplotypes are associated with differential gene expression but also differ in the Met50Val substitution (rs1047626) in ZnT9, which we show is found in homozygosis in the Denisovan genome and displays accompanying signatures suggestive of archaic introgression. Although we found no significant differences in systemic zinc content between individuals with different rs1047626 genotypes, we demonstrate that the expression of the derived isoform (ZnT9 50Val) in HEK293 cells shows a gain of function when compared with the ancestral (ZnT9 50Met) variant. Notably, the ZnT9 50Val variant was found associated with differences in zinc handling by the mitochondria and endoplasmic reticulum, with an impact on mitochondrial metabolism. Given the essential role of the mitochondria in skeletal muscle and since the derived allele at rs1047626 is known to be associated with greater susceptibility to several neuropsychiatric traits, we propose that adaptation to cold may have driven this selection event, while also impacting predisposition to neuropsychiatric disorders in modern humans

Human genetic adaptation related to cellular zinc homeostasis

SLC30A9 encodes a ubiquitously zinc transporter (ZnT9) and has been consistently suggested as a candidate for positive selection in humans. However, no direct adaptive molecular phenotype has been demonstrated. Our results provide evidence for directional selection operating in two major complementary haplotypes in Africa and East Asia. These haplotypes are associated with differential gene expression but also differ in the Met50Val substitution (rs1047626) in ZnT9, which we show is found in homozygosis in the Denisovan genome and displays accompanying signatures suggestive of archaic introgression. Although we found no significant differences in systemic zinc content between individuals with different rs1047626 genotypes, we demonstrate that the expression of the derived isoform (ZnT9 50Val) in HEK293 cells shows a gain of function when compared with the ancestral (ZnT9 50Met) variant. Notably, the ZnT9 50Val variant was found associated with differences in zinc handling by the mitochondria and endoplasmic reticulum, with an impact on mitochondrial metabolism. Given the essential role of the mitochondria in skeletal muscle and since the derived allele at rs1047626 is known to be associated with greater susceptibility to several neuropsychiatric traits, we propose that adaptation to cold may have driven this selection event, while also impacting predisposition to neuropsychiatric disorders in modern humans.This work was supported by Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI; DOI:10.13039/501100011033) with project grants RYC-2017-22227, PID2019-106232RB-I00 (to FC), PID2019-110933GB-I00 (to EB), PID2019-106755RB-I00 (to RV), and Unidad de Excelencia María de Maeztu CEX2018-000792-M (to EB and RV) and Severo Ochoa CEX2019-000910-S (to FC); and by Direcció General de Recerca, Generalitat de Catalunya with project grant 2017SGR00702 (to EB). FC was supported by Fundació Cellex, Fundació Mir-Puig, and Generalitat de Catalunya (CERCA, AGAUR). JGC was supported with an FPI-MCIN/AEI PhD contract (PRE2020-095762). GM was supported by funding from Instituto de Salud Carlos III (ISCIII) through project PI21/00612 and co-funded by the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Characterization of the structure and overexpression of ZnT9 in HEK293 cells.

(A) ZnT9 protein structure prediction by AlphaFold, showing 6 transmembrane domains and a cytosolic N-terminus, where the Met50Val substitution is indicated by a red arrow. (B) Protein sequence of the ZnT9 transporter with the Met50Val substitution in red and the predicted alpha-helices corresponding to transmembrane domains in blue. (C) Representative Western blot against HA and GAPDH in cells transfected with ZnT9-50Met, ZnT9-50Val, or an empty vector. (D) Representative pictures of the membrane and intracellular immunostaining of HEK293 cells expressing ZnT9-50Met and ZnT9-50Val isoforms (both in green). Nucleae were stained with DAPI (blue). Scale bar = 10 μm.</p

Haplotype structure of two major contrasting human haplotypes at <i>SCL30A9</i> and comparison with archaic humans.

Schematic representation of the two major human haplotypes as defined by 84 SNPs in high linkage disequilibrium (r2>0.8) with rs1047626 in CEU and CHB along the putatively inferred introgressed region (chr4: 41,977,828–42,048,441; GRCh38) and the corresponding allele states found in four high coverage Denisovan and Neanderthal genomes. Derived states are indicated in black, and ancestral alleles in white. Den to Papuan and Den to Melanesian indicate Denisovan introgression segments previously described in Melanesians [34] and Papua New Guineans [35]. The black arrow point to the rs1047626 position. GWAS hits are indicated with an asterisk (see S4 Table for details). (PDF)</p

Haplostrips distance output using four high-coverage archaic humans and the OCE, CHB, CEU and YRI populations (chr4: 41,977,828–42,048,441; GRCh38).

Haplostrips distance output using four high-coverage archaic humans and the OCE, CHB, CEU and YRI populations (chr4: 41,977,828–42,048,441; GRCh38).</p

Continental allele frequencies and GTEX data for rs1047626.

(A) Continental 1000 Genomes Project Phase 3 allele frequencies as retrieved from Ensembl (https://www.ensembl.org/index.html). (B) Multi-tissue eQTL comparison for rs1047626. (C). Differential SLC30A9 expression in brain cortex and skeletal muscle according to the rs1047626 genotypes as available at the GTEX portal (https://www.gtexportal.org/home/). NES, normalized effect sizes. (PDF)</p

Top 40 candidate variants in CEU identified by iSAFE.

Top 40 candidate variants in CEU identified by iSAFE.</p

Continental allele frequencies and GTEX data for rs1848182.

(A) Continental 1000 Genomes Project Phase 3 allele frequencies as retrieved from Ensembl (https://www.ensembl.org/index.html). (B) Multi-tissue eQTL comparison for rs1848182. (C). Differential SLC30A9 expression in the nucleus accumbens according to the rs1848182 genotypes as available at the GTEX portal (https://www.gtexportal.org/home/). NES, normalized effect sizes. (PDF)</p