[show abstract][hide abstract] ABSTRACT: Measles virus (MV), a member of the family Paramyxoviridae, remains a major cause of morbidity and mortality in the developing world. MV is spread by aerosols but the mechanism(s) responsible for the high transmissibility of MV are largely unknown. We previously infected macaques with enhanced green fluorescent protein (EGFP)-expressing recombinant (r) MV and euthanized them at a range of time points. In this study a comprehensive pathological analysis has been performed of tissues from the respiratory tract around the peak of virus replication. Isolation of virus from nose and throat swab samples showed that high levels of both cell-associated and cell-free virus were present in the upper respiratory tract. Analysis of tissue sections from lung and primary bronchus revealed localised infection of epithelial cells, concomitant infiltration of MV-infected immune cells into the epithelium and localised shedding of cells or cell debris into the lumen. While high numbers of MV-infected cells were present in the tongue, these were largely encapsulated by intact keratinocyte cell layers that likely limit virus transmission. In contrast, the integrity of tonsillar and adenoidal epithelia was disrupted with high numbers of MV-infected epithelial cells and infiltrating immune cells present throughout epithelial cell layers. Disruption was associated with large numbers of MV-infected cells or cell debris 'spilling' from epithelia into the respiratory tract. The coughing and sneezing response induced by disruption of the ciliated epithelium, leading to the expulsion of MV-infected cells, cell debris and cell-free virus, contributes to the highly infectious nature of MV.
Journal of General Virology 06/2013; · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Measles virus (MV), one of the most contagious viruses infecting humans, causes a systemic infection leading to fever, immune suppression and a characteristic maculopapular rash. However, the specific mechanism(s) responsible for the spread of MV into the respiratory epithelium in the late stages of the disease are unknown. Here we show the crucial role of PVRL4 in mediating the spread of MV from immune to epithelial cells by generating a PVRL4 'blind' recombinant wild-type MV and developing a novel in vitro co-culture model of B-cells with primary differentiated normal human bronchial epithelial cells. We utilized the macaque model of measles to analyze virus distribution in the respiratory tract prior to and at the peak of MV replication. Expression of PVRL4 was widespread in both the lower and upper respiratory tract (URT) of macaques, indicating MV transmission can be facilitated by more than only epithelial cells of the trachea. Analysis of tissues collected at early time-points after experimental MV infection demonstrated the presence of MV-infected lymphoid and myeloid cells contacting respiratory tract epithelium in the absence of infected epithelial cells, proving these immune cells seed the infection in vivo. Thereafter lateral cell-to-cell spread of MV led to the formation of large foci of infected cells in the trachea and high levels of MV infection in the URT, particularly in the nasal cavity. These novel findings have important implications for our understanding of the high transmissibility of measles.
[show abstract][hide abstract] ABSTRACT: Phocine distemper virus (PDV) was identified as the cause of a large morbillivirus outbreak among harbor seals in the North Sea in 1988. PDV is a member of the family Paramyxoviridae, genus Morbillivirus. Until now, no full-genome sequence of PDV has been available.
[show abstract][hide abstract] ABSTRACT: Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the "measles paradox". Here we show that MV preferentially infects CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.
[show abstract][hide abstract] ABSTRACT: Inactivated paramyxovirus vaccines have been associated with hypersensitivity responses upon challenge infection. For measles and canine distemper virus (CDV) safe and effective live-attenuated virus vaccines are available, but for human respiratory syncytial virus and human metapneumovirus development of such vaccines has proven difficult. We recently identified three synthetic bacterial lipopeptides that enhance paramyxovirus infections in vitro, and hypothesized these could be used as adjuvants to promote immune responses induced by live-attenuated paramyxovirus vaccines.
Here, we tested this hypothesis using a CDV vaccination and challenge model in ferrets. Three groups of six animals were intra-nasally vaccinated with recombinant (r) CDV(5804P)L(CCEGFPC) in the presence or absence of the infection-enhancing lipopeptides Pam3CSK4 or PHCSK4. The recombinant CDV vaccine virus had previously been described to be over-attenuated in ferrets. A group of six animals was mock-vaccinated as control. Six weeks after vaccination all animals were challenged with a lethal dose of rCDV strain Snyder-Hill expressing the red fluorescent protein dTomato.
Unexpectedly, intra-nasal vaccination of ferrets with rCDV(5804P)L(CCEGFPC) in the absence of lipopeptides resulted in good immune responses and protection against lethal challenge infection. However, in animals vaccinated with lipopeptide-adjuvanted virus significantly higher vaccine virus loads were detected in nasopharyngeal lavages and peripheral blood mononuclear cells. In addition, these animals developed significantly higher CDV neutralizing antibody titers compared to animals vaccinated with non-adjuvanted vaccine.
This study demonstrates that the synthetic cationic lipopeptides Pam3CSK4 and PHCSK4 not only enhance paramyxovirus infection in vitro, but also in vivo. Given the observed enhancement of immunogenicity their potential as adjuvants for other live-attenuated paramyxovirus vaccines should be considered.
[show abstract][hide abstract] ABSTRACT: Exchange of gene segments between mammalian and avian influenza A viruses may lead to the emergence of potential pandemic influenza viruses. Since co-infection of single cells with two viruses is a prerequisite for reassortment to take place, we assessed frequencies of double-infection in vitro using influenza A/H5N1 and A/H1N1 viruses expressing the reporter genes eGFP or mCherry. Double-infected A549 and Madin-Darby canine kidney cells were detected by confocal microscopy and flow cytometry.
Journal of General Virology 04/2012; 93(Pt 8):1645-8. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.
Journal of Virology 03/2011; 85(6):2695-702. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The hemagglutination inhibition (HI) assay is used most commonly for the detection of antibodies to influenza viruses. However, for the detection of antibodies to avian influenza viruses of the H5N1 subtype either induced by infection or by vaccination, the HI assay is insensitive. Therefore, the virus neutralization (VN) assay has become the method of choice to detect human serum antibodies directed to these viruses. However, this assay requires a second assay for the detection of residual virus replication, which makes it laborious to perform and less suitable for high throughput testing of large numbers of samples. Here we describe an alternative method for the detection of these antibodies, which is based on the use of reporter viruses that express the green fluorescent protein (GFP) upon infection of target cells. GFP-expressing viruses were generated carrying the HA of a variety of antigenically distinct H5N1 influenza viruses. The method proved easy to perform and could be carried out rapidly. Using a panel of antisera raised against H5N1 influenza viruses, the assay based on GFP expressing viruses was compared with the classical virus neutralization assay and the hemagglutination inhibition assay. In general, the results obtained in these assays correlated well. It was concluded that the assay based on the reporter viruses is an attractive alternative for the classical virus neutralization assay and suitable for large sero-epidemiological studies or for the assessment of vaccine efficacy in clinical trials.
[show abstract][hide abstract] ABSTRACT: The zoonotic transmissions of highly pathogenic avian influenza viruses of the H5N1 subtype that have occurred since 1997 have sparked the development of novel influenza vaccines. The advent of reverse genetics technology, cell-culture production techniques and novel adjuvants has improved the vaccine strain preparation, production process and immunogenicity of the vaccines, respectively, and has accelerated the availability of pandemic influenza vaccines. However, there is still room for improvement, and alternative vaccine preparations can be explored, such as viral vectors. Modified vaccinia virus Ankara (MVA), originally developed as a safe smallpox vaccine, can be exploited as a viral vector and has many favourable properties. Recently, we have demonstrated that an MVA-based vaccine could protect mice and macaques against infection with highly pathogenic influenza viruses of the H5N1 subtype. In the present study, recombinant MVA expressing the haemagglutinin (HA) gene of pandemic influenza A/H1N1 virus was evaluated in the ferret model. A single immunization induced modest antibody responses and afforded only modest protection against the development of severe disease upon infection with a 2009(H1N1) strain. In contrast, two immunizations induced robust antibody responses and protected ferrets from developing severe disease, confirming that MVA is an attractive influenza vaccine production platform.
Journal of General Virology 11/2010; 91(Pt 11):2745-52. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 microg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.
Journal of Virology 08/2010; 84(16):7943-52. · 5.08 Impact Factor