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Article: Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections.

Lee, Grace Sanghee / Purdy, Michael A / Choi, Youkyung

2023 Volume 13, Issue 7

Abstract: The hepatitis B virus (HBV) and hepatitis D virus (HDV) infections cause liver disease, including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV infection remains a major global health problem. In 2019, 296 million people were living with ... ...

Abstract	The hepatitis B virus (HBV) and hepatitis D virus (HDV) infections cause liver disease, including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV infection remains a major global health problem. In 2019, 296 million people were living with chronic hepatitis B and about 5% of them were co-infected with HDV. In vitro cell culture systems are instrumental in the development of therapeutic targets. Cell culture systems contribute to identifying molecular mechanisms for HBV and HDV propagation, finding drug targets for antiviral therapies, and testing antiviral agents. Current HBV therapeutics, such as nucleoside analogs, effectively suppress viral replication but are not curative. Additionally, no effective treatment for HDV infection is currently available. Therefore, there is an urgent need to develop therapies to treat both viral infections. A robust in vitro cell culture system supporting HBV and HDV infections (HBV/HDV) is a critical prerequisite to studying HBV/HDV pathogenesis, the complete life cycle of HBV/HDV infections, and consequently identifying new therapeutics. However, the lack of an efficient cell culture system hampers the development of novel antiviral strategies for HBV/HDV infections. In vitro cell culture models have evolved with significant improvements over several decades. Recently, the development of the HepG2-NTCP sec+ cell line, expressing the sodium taurocholate co-transporting polypeptide receptor (NTCP) and self-assembling co-cultured primary human hepatocytes (SACC-PHHs) has opened new perspectives for a better understanding of HBV and HDV lifecycles and the development of specific antiviral drug targets against HBV/HDV infections. We address various cell culture systems along with different cell lines and how these cell culture systems can be used to provide better tools for HBV and HDV studies.
Language	English
Publishing date	2023-07-08
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2662250-6
ISSN	2075-1729
ISSN	2075-1729
DOI	10.3390/life13071527
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Article ; Online: Natural antibody IgG levels are associated with HBeAg-positivity and seroconversion in chronic hepatitis B patients treated with entecavir.

Choi, Youkyung H / Lee, Hyun Woong / Purdy, Michael A

Scientific reports

2022 Volume 12, Issue 1, Page(s) 4382

Abstract: B1 cell-derived natural antibodies are non-specific polyreactive antibodies and can activate the complement pathway leading to lysis of enveloped virus particles before activation of the adaptive immune response. We investigated the relationship between ... ...

Abstract	B1 cell-derived natural antibodies are non-specific polyreactive antibodies and can activate the complement pathway leading to lysis of enveloped virus particles before activation of the adaptive immune response. We investigated the relationship between natural antibody levels and treatment outcomes of 126 treatment-naïve chronic hepatitis B (CHB) patients, who underwent entecavir (ETV) treatment. Serum IgG1-3 and complement C3 levels were significantly higher in HBeAg-positive patients. In pre-treatment, IgG1 (odd ratios [OR] 2.3, p < 0.0001), IgG2 (OR 9.8, p < 0.0001), IgG3 (OR 7.4, p < 0.0001), and C3 (OR 7.2, p < 0.0001) were associated with HBeAg-positive patients. At baseline, IgG2 (OR 10.2, p = 0.025), IgG4, (OR 3.4, p = 0.026), and complement C1q (OR 5.0, p = 0.0068) were associated with seroconverters. Post-treatment levels of IgG1-4 and C3/C1q were also associated with HBeAg-positive patients and seroconverters. High levels of IgG2-4 and C1q were observed in seroconverters but not in virological responders. Thus, high pretreatment and post-treatment levels of natural antibody IgG1-4, complement C3, and/or C1q were significantly associated with HBeAg-positivity and HBeAg seroconverters in CHB patients with ETV treatment. These results suggest that the presence of preexisting host immunity against chronic hepatitis B is closely related to outcome of ETV treatment.
MeSH term(s)	Antiviral Agents/therapeutic use ; Complement C1q ; Complement C3 ; DNA, Viral ; Guanine/analogs & derivatives ; Hepatitis B e Antigens ; Hepatitis B virus/genetics ; Hepatitis B, Chronic ; Humans ; Immunoglobulin G/therapeutic use ; Seroconversion ; Treatment Outcome
Chemical Substances	Antiviral Agents ; Complement C3 ; DNA, Viral ; Hepatitis B e Antigens ; Immunoglobulin G ; entecavir (5968Y6H45M) ; Guanine (5Z93L87A1R) ; Complement C1q (80295-33-6)
Language	English
Publishing date	2022-03-14
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-08457-w
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Article ; Online: Hepatitis A virus survival on drug paraphernalia.

Medrzycki, Magdalena / Kamili, Saleem / Purdy, Michael A

Journal of viral hepatitis

2020 Volume 27, Issue 12, Page(s) 1484–1494

Abstract: The ongoing hepatitis A outbreaks in multiple states in the United States have concerned public health authorities since March 2017. The outbreaks have spread throughout 30 states and include primarily persons who use drugs, including persons who inject ... ...

Abstract	The ongoing hepatitis A outbreaks in multiple states in the United States have concerned public health authorities since March 2017. The outbreaks have spread throughout 30 states and include primarily persons who use drugs, including persons who inject drugs (PWID) and persons experiencing homelessness. Contaminated drug injection paraphernalia and sharing of these items could potentially aid in transmission of hepatitis A virus (HAV) among these populations. We examined HAV survival on drug paraphernalia frequently shared among PWIDs. The effect of low pH on HAV survival using citric acid, which is frequently used by PWIDs during dose preparation, was investigated. We compared the plaque assay results with those concurrently obtained by qRT-PCR to establish whether HAV RNA levels could be used as surrogates for plaque assay results. HAV suspended in minimal essential media at room temperature infected FRhK4 cells for more than 17 weeks. HAV remained viable in syringes/needles for up to 10 weeks depending on the gauge of the needles and the syringe dead volumes, and on cookers, tourniquets and cotton balls/filter surfaces for up to 4 weeks. HAV retained its infectivity for more than 10 weeks at pH as low as 2. In conclusion, our findings show that HAV survives and remains infective in or on injection drug use equipment for 1 to 10 weeks depending on the type of paraphernalia examined and environmental conditions. These findings suggest that contaminated drug paraphernalia can potentially facilitate the transmission of HAV within populations who share these items.
MeSH term(s)	Drug Users ; Hepatitis A ; Hepatitis A virus ; Humans ; Pharmaceutical Preparations ; Substance Abuse, Intravenous ; Syringes ; United States
Chemical Substances	Pharmaceutical Preparations
Language	English
Publishing date	2020-09-14
Publishing country	England
Document type	Journal Article
ZDB-ID	1212497-7
ISSN	1365-2893 ; 1352-0504
ISSN (online)	1365-2893
ISSN	1352-0504
DOI	10.1111/jvh.13379
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Article ; Online: In vitro

Zafrullah, Mohammad / Vazquez, Carlos / Mixson-Hayden, Tonya / Purdy, Michael A

The Journal of general virology

2021 Volume 102, Issue 11

Abstract: Hepatitis B virus (HBV) infection is a global public health problem with about 257 million chronically infected people and over 887000 deaths annually. In this study, 32 whole HBV genomes of various genotypes were amplified from clinical isolates to ... ...

Abstract	Hepatitis B virus (HBV) infection is a global public health problem with about 257 million chronically infected people and over 887000 deaths annually. In this study, 32 whole HBV genomes of various genotypes were amplified from clinical isolates to create transfection clones. The clones were sequenced, and their biological properties characterized by transfecting linear HBV clones into HepG2 cells. We analysed the SPI and SPII promotor regions, X-gene, BCP/PC sequences, core, preS/S and HBV polymerase sequences. HBV clones analysed in this study revealed differential replication kinetics of viral nucleic acids and expression of proteins. Sequence analysis of HBV clones revealed mutations in preS1, preS2 and S genes; deletion and insertion and point mutations in BCP/PC region; including novel and previously reported mutations. Among the patient samples tested, HBV genotype B clones were more likely to have higher frequencies of mutations, while sub-genotype A1 and A2 clones tended to have fewer mutations. No polymerase drug resistant mutations were seen. HBeAg mutations were primarily in the BCP/PC region in genotype B, but core truncations were found in genotype E. S gene mutations affecting HBsAg expression and detection were seen in all genotypes except A2. Using an HBV clone with repetitive terminal sequences and a
MeSH term(s)	DNA, Viral/genetics ; Genetic Variation ; Genome, Viral ; Genotype ; Hepatitis B/virology ; Hepatitis B virus/genetics ; Hepatitis B virus/isolation & purification ; Humans ; Mutation ; Phylogeny ; Transfection
Chemical Substances	DNA, Viral
Language	English
Publishing date	2021-11-01
Publishing country	England
Document type	Journal Article
ZDB-ID	219316-4
ISSN	1465-2099 ; 0022-1317
ISSN (online)	1465-2099
ISSN	0022-1317
DOI	10.1099/jgv.0.001675
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Article ; Online: The effect of phylogenetic signal reduction on genotyping of hepatitis E viruses of the species Orthohepevirus A.

Purdy, Michael A / Sue, Amanda

Archives of virology

2017 Volume 162, Issue 3, Page(s) 645–656

Abstract: Commonly, hepatitis E virus (HEV) sequences are genotyped phylogenetically using subgenomic sequences. This paper examines this practice with sequences from members of the species Orthohepevirus A. As the length of sequences becomes progressively shorter, ...

Abstract	Commonly, hepatitis E virus (HEV) sequences are genotyped phylogenetically using subgenomic sequences. This paper examines this practice with sequences from members of the species Orthohepevirus A. As the length of sequences becomes progressively shorter, the number of identical sequences in an alignment tends to increase; however, these sequences retain their genotypic identity down to 100 nucleotides in length. The best substitution models tend to become less parameterized, bootstrap support decreases, and trees created from short subgenomic fragments are less likely to be isomorphic with trees from longer subgenomic fragments or complete genome sequences. However, it is still possible to correctly genotype sequences using fragments as small as 200 nucleotides. While it is possible to correctly genotype sequences with short subgenomic sequences, the estimates of evolutionary relationships between genotypes degrade to such an extent that sequences below 1600 nucleotides long cannot be used reliably to study these relationships, and comparisons of trees from different subgenomic regions with little or no sequence overlap can be problematic. Subtyping may be done, but it requires a careful examination of the region to be used to ensure that it correctly resolves the subtypes.
MeSH term(s)	Genotype ; Genotyping Techniques/methods ; Hepatitis E/virology ; Hepatitis E virus/classification ; Hepatitis E virus/genetics ; Hepatitis E virus/isolation & purification ; Humans ; Phylogeny ; RNA Viruses/classification ; RNA Viruses/genetics
Language	English
Publishing date	2017-03
Publishing country	Austria
Document type	Evaluation Studies ; Journal Article
ZDB-ID	7491-3
ISSN	1432-8798 ; 0304-8608
ISSN (online)	1432-8798
ISSN	0304-8608
DOI	10.1007/s00705-016-3135-x
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Article ; Online: Evolution of the hepatitis E virus polyproline region: order from disorder.

Purdy, Michael A

Journal of virology

2012 Volume 86, Issue 18, Page(s) 10186–10193

Abstract: The hepatitis E virus (HEV) polyproline region (PPR) is an intrinsically unstructured region (IDR). This relaxed structure allows IDRs, which are implicated in the regulation of transcription and translation, to bind multiple ligands. Originally the ... ...

Abstract	The hepatitis E virus (HEV) polyproline region (PPR) is an intrinsically unstructured region (IDR). This relaxed structure allows IDRs, which are implicated in the regulation of transcription and translation, to bind multiple ligands. Originally the nucleotide variability seen in the HEV PPR was assumed to be due to high rates of insertion and deletion. This study shows that the mutation rate is about the same in the PPR as in the rest of the nonstructural polyprotein. The difference between the PPR and the rest of the polyprotein is due to the higher tolerance of the PPR for substitutions at the first and second codon positions. With this higher promiscuity there is a shift in nucleotide occupation of these codons leading to translation of more cytosine residues: a shift that leads to more proline, alanine, serine, and threonine being encoded rather than histidine, phenylalanine, tryptophan, and tyrosine. This pattern of amino acid usage is typical of proline-rich IDRs. Increased usage of cytosine also leads to >22% of all amino acids in the PPR being prolines. Alignments of PPR sequences from HEV strains representing all genotypes indicate that all zoonotic isolates share an ancestor, and the carboxyl half of the PPR is more tolerant of mutations than the amino half. The evolution of HEV PPR, in contrast with that of the rest of the nonstructural polyprotein, is molded by pressures that lead toward increased proline usage with a corresponding decrease in the usage of aromatic amino acids, favoring formation of IDR structures.
MeSH term(s)	Animals ; Base Sequence ; Codon/genetics ; Evolution, Molecular ; Genetic Variation ; Hepatitis E virus/chemistry ; Hepatitis E virus/classification ; Hepatitis E virus/genetics ; Humans ; INDEL Mutation ; Molecular Sequence Data ; Mutation ; Peptides/chemistry ; Peptides/genetics ; RNA, Viral/genetics ; Rabbits ; Sequence Homology, Nucleic Acid ; Viral Nonstructural Proteins/chemistry ; Viral Nonstructural Proteins/genetics
Chemical Substances	Codon ; Peptides ; RNA, Viral ; Viral Nonstructural Proteins ; polyproline (25191-13-3)
Language	English
Publishing date	2012-07-18
Publishing country	United States
Document type	Journal Article
ZDB-ID	80174-4
ISSN	1098-5514 ; 0022-538X
ISSN (online)	1098-5514
ISSN	0022-538X
DOI	10.1128/JVI.01374-12
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Article: The effect of phylogenetic signal reduction on genotyping of hepatitis E viruses of the species Orthohepevirus A

Purdy, Michael A / Amanda Sue

Archives of virology. 2017 Mar., v. 162, no. 3

2017

Abstract	Commonly, hepatitis E virus (HEV) sequences are genotyped phylogenetically using subgenomic sequences. This paper examines this practice with sequences from members of the species Orthohepevirus A. As the length of sequences becomes progressively shorter, the number of identical sequences in an alignment tends to increase; however, these sequences retain their genotypic identity down to 100 nucleotides in length. The best substitution models tend to become less parameterized, bootstrap support decreases, and trees created from short subgenomic fragments are less likely to be isomorphic with trees from longer subgenomic fragments or complete genome sequences. However, it is still possible to correctly genotype sequences using fragments as small as 200 nucleotides. While it is possible to correctly genotype sequences with short subgenomic sequences, the estimates of evolutionary relationships between genotypes degrade to such an extent that sequences below 1600 nucleotides long cannot be used reliably to study these relationships, and comparisons of trees from different subgenomic regions with little or no sequence overlap can be problematic. Subtyping may be done, but it requires a careful examination of the region to be used to ensure that it correctly resolves the subtypes.
Keywords	Hepatitis E virus ; genotype ; genotyping ; models ; nucleotide sequences ; nucleotides ; phylogeny
Language	English
Dates of publication	2017-03
Size	p. 645-656.
Publishing place	Springer Vienna
Document type	Article
ZDB-ID	7491-3
ISSN	1432-8798 ; 0304-8608
ISSN (online)	1432-8798
ISSN	0304-8608
DOI	10.1007/s00705-016-3135-x
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Article ; Online: Transcriptome analysis in rhesus macaques infected with hepatitis E virus genotype 1/3 infections and genotype 1 re-infection.

Choi, Youkyung H / Zhang, Xiugen / Srinivasamoorthy, Ganesh / Purdy, Michael A

PloS one

2020 Volume 15, Issue 9, Page(s) e0237618

Abstract: Hepatitis E virus (HEV) genotype 1 (gt1) and gt3 infections have distinct epidemiologic characteristics and genotype-specific molecular mechanisms of pathogenesis are not well characterized. Previously, we showed differences in immune response-related ... ...

Abstract	Hepatitis E virus (HEV) genotype 1 (gt1) and gt3 infections have distinct epidemiologic characteristics and genotype-specific molecular mechanisms of pathogenesis are not well characterized. Previously, we showed differences in immune response-related gene expression profiles of HEV gt1 and gt3 infections using qPCR. We hypothesize that HEV gt1 and gt3 infections induce transcriptome modifications contributing to disease pathogenesis. RNAseq analysis was performed using liver biopsy samples of naïve (baseline), HEV gt1, or gt3-infected rhesus macaques, and nine anti-HEV positive rhesus macaques re-inoculated with HEV gt1. All 10 primary HEV gt1/gt3 infected animals exhibited the typical course of acute viral hepatitis and cleared the infection between 27 to 67 days after inoculation. Viremic stages of HEV infection were defined as early, peak, and decline based on HEV RNA titers in daily stool specimens. During early, peak, and decline phases of infection, HEV gt1 induced 415, 417, and 1769 differentially expressed genes, respectively, and 310, 678, and 388 genes were differentially expressed by HEV gt3, respectively (fold change ≥ 2.0, p-value ≤ 0.05). In the HEV gt1 infection, genes related to metabolic pathways were differentially expressed during the three phases of infection. In contrast, oxidative reduction (early phase), immune responses (peak phase), and T cell cytokine production (decline phase) were found to be regulated during HEV gt3 infection. In addition, FoxO and MAPK signaling pathways were differentially regulated in re-infected and protected animals against HEV gt1 reinfection, respectively. Significant differences of hepatic gene regulation exist between HEV gt1 and gt3 infections. These findings reveal a new link between molecular pathogenesis and epidemiological characteristics seen in HEV gt1 and gt3 infections.
MeSH term(s)	Animals ; Biopsy ; Gene Expression Profiling ; Gene Ontology ; Genotype ; Hepatitis E/veterinary ; Hepatitis E virus/genetics ; Liver/pathology ; Macaca mulatta/virology ; Sequence Analysis, RNA
Language	English
Publishing date	2020-09-02
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, P.H.S.
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0237618
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Article: Hepatitis B virus S gene escape mutants.

Purdy, Michael A

Asian journal of transfusion science

2011 Volume 1, Issue 2, Page(s) 62–70

Abstract: Hepatitis B virus (HBV) can be classified into nine immunological subtypes or eight genotypes. The most prevalent genotypes in Asia are genotypes B and C. HBV is transmitted parenteraly and can produce either asymptomatic or symptomatic disease. Although ...

Abstract	Hepatitis B virus (HBV) can be classified into nine immunological subtypes or eight genotypes. The most prevalent genotypes in Asia are genotypes B and C. HBV is transmitted parenteraly and can produce either asymptomatic or symptomatic disease. Although the consequences of acute hepatitis B can be severe, serious sequelae are associated with chronic infections. HBV seroprevalence ranges from intermediate (2%-7%) to high (≥8%) levels in Asia. Several strategies for the control and prevention of HBV infection have been found to be efficacious. They include vaccination and the administration of HBIG, interferon-a and nucleoside/nucleotide analogues. However, these procedures also apply selective pressures on HBV in infected individuals leading to the generation and accumulation of mutations in the S gene. Most of these mutations occur in the major hydrophilic region (MHR) of the S gene. These mutations create public health concerns as they can be responsible for reactivation of hepatitis B and occult hepatitis B infection. The inability to detect occult infections means that these individuals may become blood donors. This suggests that new strategies for donor evaluation and selection may need to be developed to protect the blood supply.
Language	English
Publishing date	2011-09-06
Publishing country	India
Document type	Journal Article
ZDB-ID	2267310-6
ISSN	1998-3565 ; 0973-6247
ISSN (online)	1998-3565
ISSN	0973-6247
DOI	10.4103/0973-6247.33445
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Article ; Online: Hepatitis B vaccine delivered by microneedle patch: Immunogenicity in mice and rhesus macaques.

Choi, Youkyung / Lee, Grace Sanghee / Li, Song / Lee, Jeong Woo / Mixson-Hayden, Tonya / Woo, Jungreem / Xia, Dengning / Prausnitz, Mark R / Kamili, Saleem / Purdy, Michael A / Tohme, Rania A

Vaccine

2023 Volume 41, Issue 24, Page(s) 3663–3672

Abstract: Vaccination against hepatitis B using a dissolving microneedle patch (dMNP) could increase access to the birth dose by reducing expertise needed for vaccine administration, refrigerated storage, and safe disposal of biohazardous sharps waste. In this ... ...

Abstract	Vaccination against hepatitis B using a dissolving microneedle patch (dMNP) could increase access to the birth dose by reducing expertise needed for vaccine administration, refrigerated storage, and safe disposal of biohazardous sharps waste. In this study, we developed a dMNP to administer hepatitis B surface antigen (HBsAg) adjuvant-free monovalent vaccine (AFV) at doses of 5 µg, 10 µg, and 20 µg, and compared its immunogenicity to vaccination with 10 µg of standard monovalent HBsAg delivered by intramuscular (IM) injection either in an AFV format or as aluminum-adjuvanted vaccine (AAV). Vaccination was performed on a three dose schedule of 0, 3, and 9 weeks in mice and 0, 4, and 24 weeks in rhesus macaques. Vaccination by dMNP induced protective levels of anti-HBs antibody responses (≥10 mIU/ml) in mice and rhesus macaques at all three HBsAg doses studied. HBsAg delivered by dMNP induced higher anti-HBsAg antibody (anti-HBs) responses than the 10 µg IM AFV, but lower responses than 10 µg IM AAV, in mice and rhesus macaques. HBsAg-specific CD4+ and CD8+ T cell responses were detected in all vaccine groups. Furthermore, we analyzed differential gene expression profiles related to each vaccine delivery group and found that tissue stress, T cell receptor signaling, and NFκB signaling pathways were activated in all groups. These results suggest that HBsAg delivered by dMNP, IM AFV, and IM AAV have similar signaling pathways to induce innate and adaptive immune responses. We further demonstrated that dMNP was stable at room temperature (20 °C-25 °C) for 6 months, maintaining 67 ± 6 % HBsAg potency. This study provides evidence that delivery of 10 µg (birth dose) AFV by dMNP induced protective levels of antibody responses in mice and rhesus macaques. The dMNPs developed in this study could be used to improve hepatitis B birth dose vaccination coverage levels in resource limited regions to achieve and maintain hepatitis B elimination.
MeSH term(s)	Animals ; Mice ; Hepatitis B Vaccines ; Macaca mulatta ; Hepatitis B Surface Antigens ; Vaccination/methods ; Hepatitis B Antibodies ; Hepatitis B/prevention & control ; Adjuvants, Immunologic
Chemical Substances	Hepatitis B Vaccines ; Hepatitis B Surface Antigens ; Hepatitis B Antibodies ; Adjuvants, Immunologic
Language	English
Publishing date	2023-05-11
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	605674-x
ISSN	1873-2518 ; 0264-410X
ISSN (online)	1873-2518
ISSN	0264-410X
DOI	10.1016/j.vaccine.2023.05.005
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