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| 4. |
- Adams, WC, et al.
(författare)
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Adenovirus serotype 5 infects human dendritic cells via a coxsackievirus-adenovirus receptor-independent receptor pathway mediated by lactoferrin and DC-SIGN
- 2009
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Ingår i: The Journal of general virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 90:Pt 7, s. 1600-1610
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Tidskriftsartikel (refereegranskat)abstract
- The coxsackievirus–adenovirus receptor (CAR) is the described primary receptor for adenovirus serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression. Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of these cells. Lactoferrin (Lf), an abundant protein in many bodily fluids known for its antiviral and antibacterial properties, promoted rAd5 infection in all cell populations except plasmacytoid DCs using a CAR-independent process. Lf caused phenotypic differentiation of the DCs, but cell activation played only a minor role in the increase in infection frequencies. The C-type lectin receptor DC-SIGN facilitated viral entry of rAd5–Lf complexes and this was dependent on high-mannose-type N-linked glycans on Lf. These results suggest that Lf present at high levels at mucosal sites can facilitate rAd5 attachment and enhance infection of DCs. A better understanding of the tropism and receptor mechanisms of Ad5 may help explain Ad5 pathogenesis and guide the engineering of improved rAd vectors.
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| 5. |
- Aljedani, SS, et al.
(författare)
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Structurally related but genetically unrelated antibody lineages converge on an immunodominant HIV-1 Env neutralizing determinant following trimer immunization
- 2021
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Ingår i: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374. ; 17:9, s. e1009543-
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.
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| 13. |
- Chernyshev, M, et al.
(författare)
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VDJ Gene Usage in IgM Repertoires of Rhesus and Cynomolgus Macaques
- 2022
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Ingår i: Frontiers in immunology. - : Frontiers Media SA. - 1664-3224. ; 12, s. 815680-
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Tidskriftsartikel (refereegranskat)abstract
- Macaques are frequently used to evaluate candidate vaccines and to study infection-induced antibody responses, requiring an improved understanding of their naïve immunoglobulin (IG) repertoires. Baseline gene usage frequencies contextualize studies of antigen-specific immune responses, providing information about how easily one may stimulate a response with a particular VDJ recombination. Studies of human IgM repertoires have shown that IG VDJ gene frequencies vary several orders of magnitude between the most and least utilized genes in a manner that is consistent across many individuals but to date similar analyses are lacking for macaque IgM repertoires. Here, we quantified VDJ gene usage levels in unmutated IgM repertoires of 45 macaques, belonging to two species and four commonly used subgroups: Indian and Chinese origin rhesus macaques and Indonesian and Mauritian origin cynomolgus macaques. We show that VDJ gene frequencies differed greatly between the most and least used genes, with similar overall patterns observed in macaque subgroups and individuals. However, there were also clear differences affecting the use of specific V, D and J genes. Furthermore, in contrast to humans, macaques of both species utilized IGHV4 family genes to a much higher extent and showed evidence of evolutionary expansion of genes of this family. Finally, we used the results to inform the analysis of a broadly neutralizing HIV-1 antibody elicited in SHIV-infected rhesus macaques, RHA1.V2.01, which binds the apex of the Env trimer in a manner that mimics the binding mode of PGT145. We discuss the likelihood that similar antibodies could be elicited in different macaque subgroups.
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| 16. |
- Dai, KF, et al.
(författare)
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HIV-1 Vaccine-elicited Antibodies Reverted to Their Inferred Naive Germline Reveal Associations between Binding Affinity and in vivo Activation
- 2016
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6, s. 20987-
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Tidskriftsartikel (refereegranskat)abstract
- The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) vaccination is exceedingly difficult. Suboptimal engagement of naïve B cells is suggested to limit these low frequency events, especially at the conserved CD4bs. Here, we analyzed CD4bs-directed monoclonal antibodies (mAbs) elicited by YU2 gp140-foldon trimers in a non-human primate by selective sorting using CD4bs “knock out” trimers. Following two inoculations, the CD4bs-directed mAbs efficiently recognized the eliciting immunogen in their affinity-maturing state but did not recognize CD4bs-defective probes. We reverted these mAbs to their most likely inferred germline (igL) state, leaving the HCDR3 unaltered, to establish correlates of in vitro affinity to in vivo activation. Most igL-reverted mAbs bound the eliciting gp140 immunogen, indicating that CD4bs-directed B cells possessing reasonable affinity existed in the naïve repertoire. We detected relatively high affinities for the majority of the igL mAbs to gp120 and of Fabs to gp140, which, as expected, increased when the antibodies ‘matured’ following vaccination. Affinity increases were associated with slower off-rates as well as with acquisition of neutralizing capacity. These data reveal in vitro binding properties associated with in vivo activation that result in functional archiving of antigen-specific B cells elicited by a complex glycoprotein antigen following immunization.
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- Forsell, MNE, et al.
(författare)
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Increased human immunodeficiency virus type 1 Env expression and antibody induction using an enhanced alphavirus vector
- 2007
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Ingår i: The Journal of general virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 88:Pt 10, s. 2774-2779
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Tidskriftsartikel (refereegranskat)abstract
- Viral vectors encoding heterologous vaccine antigens are potent inducers of cellular immune responses, but they are generally less efficient at stimulating humoral immunity. To improve the induction of antibody responses by Semliki Forest virus-based vaccines, a vector encoding a translation-enhancer element and a novel internal signal sequence for increased expression and secretion of soluble antigens was designed. Approximately tenfold more human immunodeficiency virus type 1 gp120 was secreted into culture supernatants of infected cells using the enhanced vector compared with the parental vector. This translated into a significant increase in gp120-specific antibodies in immunized mice, suggesting that antigen-expression levels from the parental vector are limiting for induction of antibody responses. These data encourage the use of the enhanced vector for elicitation of immune responses against heterologous antigens during vaccination.
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| 36. |
- Hanke, L, et al.
(författare)
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An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction
- 2020
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1, s. 4420-
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Tidskriftsartikel (refereegranskat)abstract
- SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.
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| 37. |
- Hardt, U, et al.
(författare)
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Analysis of IGH allele content in a sample group of rheumatoid arthritis patients demonstrates unrevealed population heterogeneity
- 2023
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Ingår i: Frontiers in immunology. - : Frontiers Media SA. - 1664-3224. ; 14, s. 1073414-
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Tidskriftsartikel (refereegranskat)abstract
- Immunoglobulin heavy chain (IGH) germline gene variations influence the B cell receptor repertoire, with resulting biological consequences such as shaping our response to infections and altering disease susceptibilities. However, the lack of information on polymorphism frequencies in the IGH loci at the population level makes association studies challenging. Here, we genotyped a pilot group of 30 individuals with rheumatoid arthritis (RA) to examine IGH allele content and frequencies in this group. Eight novel IGHV alleles and one novel IGHJ allele were identified in the study. 15 cases were haplotypable using heterozygous IGHJ6 or IGHD anchors. One variant, IGHV4-34*01_S0742, was found in three out of 30 cases and included a single nucleotide change resulting in a non-canonical recombination signal sequence (RSS) heptamer. This variant allele, shown by haplotype analysis to be non-expressed, was also found in three out of 30 healthy controls and matched a single nucleotide polymorphism (SNP) described in the 1000 Genomes Project (1KGP) collection with frequencies that varied between population groups. Our finding of previously unreported alleles in a relatively small group of individuals with RA illustrates the need for baseline information about IG allelic frequencies in targeted study groups in preparation for future analysis of these genes in disease association studies.
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| 44. |
- Kaduk, M, et al.
(författare)
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Addressing IGHV Gene Structural Diversity Enhances Immunoglobulin Repertoire Analysis: Lessons From Rhesus Macaque
- 2022
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Ingår i: Frontiers in immunology. - : Frontiers Media SA. - 1664-3224. ; 13, s. 818440-
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Tidskriftsartikel (refereegranskat)abstract
- The accurate germline gene assignment and assessment of somatic hypermutation in antibodies induced by immunization or infection are important in immunological studies. Here, we illustrate issues specific to the construction of comprehensive immunoglobulin (IG) germline gene reference databases for outbred animal species using rhesus macaques, a frequently used non-human primate model, as a model test case. We demonstrate that the genotypic variation found in macaque germline inference studies is reflected in similar levels of gene diversity in genomic assemblies. We show that the high frequency of IG heavy chain V (IGHV) region structural and gene copy number variation between subjects means that individual animals lack genes that are present in other animals. Therefore, gene databases compiled from a single or too few animals will inevitably result in inaccurate gene assignment and erroneous SHM level assessment for those genes it lacks. We demonstrate this by assigning a test macaque IgG library to the KIMDB, a database compiled of germline IGHV sequences from 27 rhesus macaques, and, alternatively, to the IMGT rhesus macaque database, based on IGHV genes inferred primarily from the genomic sequence of the rheMac10 reference assembly, supplemented with 10 genes from the Mmul_051212 assembly. We found that the use of a gene-restricted database led to overestimations of SHM by up to 5% due to misassignments. The principles described in the current study provide a model for the creation of comprehensive immunoglobulin reference databases from outbred species to ensure accurate gene assignment, lineage tracing and SHM calculations.
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| 47. |
- Khoenkhoen, S, et al.
(författare)
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IκBNS expression in B cells is dispensable for IgG responses to T cell-dependent antigens
- 2022
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Ingår i: Frontiers in immunology. - : Frontiers Media SA. - 1664-3224. ; 13, s. 1000755-
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Tidskriftsartikel (refereegranskat)abstract
- Mice lacking the atypical inhibitory kappa B (IκB) protein, IκBNS, a regulator of the NF-κB pathway encoded by the nfkbid gene, display impaired antibody responses to both T cell-independent (TI) and T cell-dependent (TD) antigens. To better understand the basis of these defects, we crossed mice carrying floxed nfkbid alleles with mice expressing Cre under the transcriptional control of the Cd79a gene to create mice that lacked IκBNS expression only in B cells. Analyses of these conditional knock-out mice revealed intact CD4+ and CD8+ T cell populations, including preserved frequencies of FoxP3+ regulatory T cells, which are known to be reduced in IκBNS knock-out mice. Like IκBNS knock-out mice, mice with conditional IκBNS ablation in B cells displayed defective IgM responses to TI antigens and a severe reduction in peritoneal B-1a cells. However, in contrast to mice lacking IκBNS altogether, the conditional IκBNS knock-out mice responded well to TD antigens compared to the control mice, with potent IgG responses following immunization with the viral antigen, rSFV-βGal or the widely used hapten-protein model antigen, NP-CGG. Furthermore, B cell intrinsic IκBNS expression was dispensable for germinal center (GC) formation and T follicular helper cell responses to NP-CGG immunization. The results presented here suggest that the defect in antibody responses to TD antigens observed in IκBNS knock-out mice results from a B cell extrinsic defect.
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| 48. |
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| 49. |
- Klein, F, et al.
(författare)
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Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants
- 2014
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Ingår i: The Journal of experimental medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 211:12, s. 2361-2372
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian–human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants.
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