| 1. |
- Hinkula, Jorma, et al.
(författare)
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Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice
- 2017
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Ingår i: Journal of Virology. - : AMER SOC MICROBIOLOGY. - 0022-538X .- 1098-5514. ; 91:10
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR(- / -)) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDAapproved vaccine, and there are still gaps in our knowledge of the immune responses to infection. The recently developed mouse models mimic human CCHF disease and are useful to study the immunogenicity and the protection by vaccine candidates. Our study shows that mice vaccinated with a specific DNA vaccine were fully protected. Importantly, we show that neutralizing antibodies are not sufficient for protection against CCHFV challenge but that an extra Th1-specific cellular response is required. Moreover, we describe the identification of five conserved B-cell epitopes, of which only one was previously known, that could be of great importance for the development of diagnostics tools and the improvement of vaccine candidates.
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| 2. |
- Krzywkowski, Tomasz, et al.
(författare)
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Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection
- 2017
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Ingår i: Journal of Virology. - : AMER SOC MICROBIOLOGY. - 0022-538X .- 1098-5514. ; 91:11
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Tidskriftsartikel (refereegranskat)abstract
- An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.
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| 3. |
- Sharma, Sumit, et al.
(författare)
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Human Sera Collected between 1979 and 2010 Possess Blocking-Antibody Titers to Pandemic GII.4 Noroviruses Isolated over Three Decades
- 2017
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Ingår i: Journal of Virology. - : AMER SOC MICROBIOLOGY. - 0022-538X .- 1098-5514. ; 91:14
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of pandemic GII.4 norovirus (NoV) strains has been proposed to occur due to changes in receptor usage and thereby to lead to immune evasion. To address this hypothesis, we measured the ability of human sera collected between 1979 and 2010 to block glycan binding of four pandemic GII. 4 noroviruses isolated in the last 4 decades. In total, 268 sera were investigated for 50% blocking titer (BT50) values of virus-like particles (VLPs) against pig gastric mucin (PGM) using 4 VLPs that represent different GII. 4 norovirus variants identified between 1987 and 2012. Pre- and postpandemic sera (sera collected before and after isolation of the reference NoV strain) efficiently prevented binding of VLP strains MD145 (1987), Grimsby (1995), and Houston (2002), but not the Sydney (2012) strain, to PGM. No statistically significant difference in virus-blocking titers was observed between pre- and postpandemic sera. Moreover, paired sera showed that blocking titers of amp;gt;= 160 were maintained over a 6-year period against MD145, Grimsby, and Houston VLPs. Significantly higher serum blocking titers (geometric mean titer [GMT], 1,704) were found among IgA-deficient individuals than among healthy blood donors (GMT, 90.9) (P amp;lt; 0.0001). The observation that prepandemic sera possess robust blocking capacity for viruses identified decades later suggests a common attachment factor, at least until 2002. Our results indicate that serum IgG possesses antibody-blocking capacity and that blocking titers can be maintained for at least 6 years against 3 decades of pandemic GII. 4 NoV. IMPORTANCE Human noroviruses (NoVs) are the major cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) in saliva and gut recognize NoV and are the proposed ligands that facilitate infection. Polymorphisms in HBGA genes, and in particular a nonsense mutation in FUT2 (G428A), result in resistance to global dominating GII. 4 NoV. The emergence of new pandemic GII. 4 strains occurs at intervals of several years and is proposed to be attributable to epochal evolution, including amino acid changes and immune evasion. However, it remains unclear whether exposure to a previous pandemic strain stimulates immunity to a pandemic strain identified decades later. We found that prepandemic sera possess robust virus-blocking capacity against viruses identified several decades later. We also show that serum lacking IgA antibodies is sufficient to block NoV VLP binding to HBGAs. This is essential, considering that 1 in every 600 Caucasian children is IgA deficient.
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| 4. |
- Sharma, S., et al.
(författare)
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Human Sera Collected between 1979 and 2010 Possess Blocking-Antibody Titers to Pandemic GII.4 Noroviruses Isolated over Three Decades
- 2017
-
Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 91:14
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of pandemic GII.4 norovirus (NoV) strains has been proposed to occur due to changes in receptor usage and thereby to lead to immune evasion. To address this hypothesis, we measured the ability of human sera collected between 1979 and 2010 to block glycan binding of four pandemic GII. 4 noroviruses isolated in the last 4 decades. In total, 268 sera were investigated for 50% blocking titer (BT50) values of virus-like particles (VLPs) against pig gastric mucin (PGM) using 4 VLPs that represent different GII. 4 norovirus variants identified between 1987 and 2012. Pre- and postpandemic sera (sera collected before and after isolation of the reference NoV strain) efficiently prevented binding of VLP strains MD145 (1987), Grimsby (1995), and Houston (2002), but not the Sydney (2012) strain, to PGM. No statistically significant difference in virus-blocking titers was observed between pre- and postpandemic sera. Moreover, paired sera showed that blocking titers of >= 160 were maintained over a 6-year period against MD145, Grimsby, and Houston VLPs. Significantly higher serum blocking titers (geometric mean titer [GMT], 1,704) were found among IgA-deficient individuals than among healthy blood donors (GMT, 90.9) (P < 0.0001). The observation that prepandemic sera possess robust blocking capacity for viruses identified decades later suggests a common attachment factor, at least until 2002. Our results indicate that serum IgG possesses antibody-blocking capacity and that blocking titers can be maintained for at least 6 years against 3 decades of pandemic GII. 4 NoV. IMPORTANCE Human noroviruses (NoVs) are the major cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) in saliva and gut recognize NoV and are the proposed ligands that facilitate infection. Polymorphisms in HBGA genes, and in particular a nonsense mutation in FUT2 (G428A), result in resistance to global dominating GII. 4 NoV. The emergence of new pandemic GII. 4 strains occurs at intervals of several years and is proposed to be attributable to epochal evolution, including amino acid changes and immune evasion. However, it remains unclear whether exposure to a previous pandemic strain stimulates immunity to a pandemic strain identified decades later. We found that prepandemic sera possess robust virus-blocking capacity against viruses identified several decades later. We also show that serum lacking IgA antibodies is sufficient to block NoV VLP binding to HBGAs. This is essential, considering that 1 in every 600 Caucasian children is IgA deficient.
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| 5. |
- Storm, Rickard J, et al.
(författare)
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Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
- 2017
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 91:5
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Tidskriftsartikel (refereegranskat)abstract
- Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVβ3, αVβ5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVβ5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, β1, and β4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, β3, or β5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVβ1 and α3β1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and β1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVβ5 in order to enter nonocular human cells. In this study, we found that αVβ5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVβ1 and α3β1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.
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