Judith H. Aberle

Single versus dual respiratory virus infections in hospitalized infants : Impact on clinical course of disease and interferon-γ response

Background: Dual respiratory viral infections are frequently associated with lower respiratory tract illness in infants. This study aimed to determine the impact of a dual respiratory viral infection on specific aspects of the infants immune response and the clinical course of illness. Methods: A prospective study was performed with 772 infants hospitalized from October 2000 through July 2004. Sensitive polymerase chain reaction methodology revealed the presence of a single respiratory virus in 443 (57%) of 772 cases, whereas dual infections were identified in 153 (20%) of cases. From 250 infants with confirmed respiratory viral infection, fresh heparinized blood was analyzed for interferon-γ (IFN-γ) responses by flow cytometry. Of these, 191 patients had a single infection with respiratory syncytial virus (RSV), rhinoviruses, adenoviruses or influenza viruses; and 59 patients had a dual infection with RSV and rhinoviruses, RSV and adenoviruses, influenza viruses and rhinoviruses or adenoviruses and rhinoviruses. The clinical features and peripheral lymphocyte IFN-γ responses were compared among infants with single or dual infections. Results: It was found that dual infections with non-RSV respiratory viruses induced peripheral blood mononuclear cell IFN-γ responses that mimic those of single infections, whereas coinfection with RSV was associated with reduced IFN-γ responses and a more severe clinical course of lower respiratory tract disease. Conclusions: The results indicate that the clinical characteristics and the IFN-γ response differ significantly in single and dual respiratory viral infection, depending on the nature of the simultaneously detected viruses. In dual infections, RSV involvement was associated with a decreased IFN-γ response in peripheral blood mononuclear cell and an increase in severity of illness.

Effectiveness of Reverse Transcription-PCR, Virus Isolation, and Enzyme-Linked Immunosorbent Assay for Diagnosis of Influenza A Virus Infection in Different Age Groups

ABSTRACT The degrees of effectiveness of reverse transcription (RT)-PCR, virus isolation, and antigen enzyme-linked immunosorbent assay (ELISA) for the detection of influenza A virus were evaluated with nasopharyngeal swabs from 150 patients (1 week to 86 years old) with influenza A virus infection. RT-PCR had a sensitivity for influenza A virus in stock virus preparations 103 times higher than virus isolation and 106 to 107 times higher than ELISA. The detection rate achieved by RT-PCR in clinical samples was clearly higher (93%) than that by virus isolation (80%) and ELISA (62%). Despite low overall detection rates achieved by antigen ELISA, samples from patients younger than 5 years old yielded higher-than-average rates in this rapid assay (88%). The likelihood of negative results in the ELISA increased significantly with increasing age of the patient (P < 0.01). The degrees of effectiveness of RT-PCR and virus isolation were not influenced by the age of the patient. Neither influenza immunizations nor the interval between onset of symptoms and laboratory investigation (mean, 4.7 days; standard deviation, 3.3 days) affected results obtained by the three test systems. Our results demonstrate that the ELISA is reliable for rapid laboratory diagnosis of influenza in infants and young children, but for older patients application of RT-PCR or virus isolation is necessary to avoid false negative results.

In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model.

Live virus vaccines have in many cases proven to be an extremely effective tool for the prevention of viral diseases. However, the production of conventional live vaccines in eukaryotic cell cultures has many disadvantages, including the potential for contamination with adventitious agents and genetic alterations during propagation, making it necessary to do extensive testing before distribution. Based on results obtained with a flavivirus (tick-borne encephalitis virus) in an experimental animal system, we propose a novel live attenuated virus vaccination strategy consisting of the application of in vitro-synthesized infectious RNA instead of the live virus itself. When administered using the GeneGun, less than 1 ng of RNA was required to initiate replication of virus that was attenuated by a specifically engineered deletion and this induced a protective immunity in laboratory mice. Because this approach uses RNA, it does not have the potential drawbacks of DNA vaccines and thus combines the advantages of conventional live virus vaccines (for example, mimicking natural infection and inducing long-lasting immunity) with those of nucleic acid-based vaccines (for example, ease of production without a requirement for eukaryotic cell culture, stability and purity).

Mimicking live flavivirus immunization with a noninfectious RNA vaccine

Flaviviruses are human pathogens of world-wide medical importance. They have recently received much additional attention because of their spread to new regions (such as West Nile virus to North America), highlighting their potential as newly emerging disease agents. Using tick-borne encephalitis virus, we have developed and evaluated in mice a new genetic vaccine based on self-replicating but noninfectious RNA. This RNA contains all of the necessary genetic information for establishing its replication machinery in the host cell, thus mimicking a natural infection. However, genetic modifications in the region encoding the capsid protein simultaneously prevent the assembly of infectious virus particles and promote the secretion of noninfectious subviral particles that elicit neutralizing antibodies. These characteristics demonstrate that a new generation of flavivirus vaccines can be designed that stimulate the same spectrum of innate and specific immune responses as a live vaccine but have the safety features of an inactivated vaccine.

Serum Level of IP-10 Increases Predictive Value of IL28B Polymorphisms for Spontaneous Clearance of Acute HCV Infection

BACKGROUND & AIMS Single nucleotide polymorphisms (SNPs) in IL28B and serum levels of interferon γ inducible protein 10 (IP-10) predict outcomes of antiviral therapy in patients with chronic hepatitis C. We associated IL28B SNPs rs12979860 and rs8099917, along with serum levels of IP-10, with outcomes of patients with acute hepatitis C (AHC). METHODS We studied 120 patients with AHC (64 male; 37 ± 16 years old) and 96 healthy individuals (controls). The IL28B SNPs rs12979860 and rs8099917 were detected using real-time polymerase chain reaction; serum concentrations of IP-10 were measured by enzyme-linked immunosorbent assays of 62 patients with AHC. RESULTS Hepatitis C virus was cleared spontaneously from 59 patients (49.2%). The IL28B rs12979860 C/C genotype was more frequent among patients with AHC than controls (62.5% vs 39.6%; P < .001) and among patients with spontaneous clearance than those without (74.6% vs 51.7%; P = .02) (positive predictive value, 60.3%). Patients with IL28B rs12979860 C/C more frequently developed jaundice (53.2% vs 27.6%; P = .022) than carriers of the T allele. The median level of IP-10 was lower among patients with AHC and spontaneous clearance (764 [113-2470] pg/mL) than those without spontaneous clearance (1481 [141-4412] pg/mL; P = .006). Based on receiver operating characteristic analysis, 540 pg/mL IP-10 was set as the cutoff for patients most likely to have spontaneous clearance (positive predictive value, 71.4%; negative predictive value, 65.9%). Including data on IP-10 levels increased the ability of the IL28B rs12979860 C/C to identify patients most likely to have spontaneous clearance (83% of those who had an IP-10 level <540 pg/mL and 32% who had an IP-10 level >540 pg/mL) (P < .01). CONCLUSIONS The combination of serum level of IP-10 and SNPs in IL28B can identify patients with AHC who are most likely to undergo spontaneous clearance and those in need of early antiviral therapy.

Quantitative real time PCR detection of Varicella-zoster virus DNA in cerebrospinal fluid in patients with neurological disease

Varicella-zoster virus (VZV) reactivation can lead to the development of neurological disease. Diagnosis has been based on the detection of VZV DNA in cerebrospinal fluid (CSF) by PCR-based methods. The aim of this study was to determine whether the VZV DNA copy number in the CSF correlates with the course of the disease and to determine its prognostic relevance. VZV DNA was quantified in CSF samples obtained from 30 patients with neurological disease due to VZV reactivation using real time PCR, and the VZV DNA copy number was correlated to the clinical and laboratory findings for each case. Viral loads ranged from 50 copies/ml to 2.6×108 copies/ml. Significantly higher viral loads [geometric mean (GM): 7.2 x 104 copies/ml] were found in patients with encephalitis compared to patients with meningitis (GM: 4.1×103 copies/ml) (P=0.01, Mann-Whitney U test). In eight patients without zoster dermal lesions no significant difference in viral load (GM: 4.6×103) was detected compared to patients exhibiting dermal lesions (GM: 2.2×104) (P=0.14). High copy numbers of VZV DNA in CSF were clearly associated with the severity of neurological disease and none of the patients with a VZV viral load below 104 copies had a disease course which required intensive care.

Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus.

ABSTRACT A new vaccination principle against flaviviruses, based on a tick-borne encephalitis virus (TBEV) self-replicating noninfectious RNA vaccine that produces subviral particles, has recently been introduced (R. M. Kofler, J. H. Aberle, S. W. Aberle, S. L. Allison, F. X. Heinz, and C. W. Mandl, Proc. Natl. Acad. Sci. USA 7:1951-1956, 2004). In this study, we evaluated the potential of the self-replicating RNA vaccine in mice in comparison to those of live, attenuated vaccines and a formalin-inactivated whole-virus vaccine (ImmunInject). For this purpose, mice were immunized using gene gun-mediated application of the RNA vaccine and tested for CD8+ T-cell responses, long-term duration, neutralizing capacity, and isotype profile of specific antibodies and protection against lethal virus challenge. We demonstrate that the self-replicating RNA vaccine induced a broad-based, humoral and cellular (Th1 and CD8+ T-cell response) immune response comparable to that induced by live vaccines and that it protected mice from challenge. Even a single immunization with 1 μg of the replicon induced a long-lasting antibody response, characterized by high neutralizing antibody titers, which were sustained for at least 1 year. Nevertheless, it was possible to boost this response further by a second injection with the RNA vaccine, even in the presence of a concomitant CD8+ T-cell response. In this way it was possible to induce a balanced humoral and cellular immune response, similar to infection-induced immunity but without the safety hazards of infectious agents. The results also demonstrate the value of TBEV replicon RNA for inducing protective long-lasting antiviral responses.

Human metapneumovirus subgroup changes and seasonality during epidemics.

Background: Human metapneumovirus (HMPV) is a major cause of respiratory tract illness in young children and causes annual outbreaks in winter and spring seasons. We evaluated the subgroups of HMPV that caused annual outbreaks and its seasonal occurrence during a 21-year period. Methods: Real-time PCR was used for detection of HMPV in 3576 nasopharyngeal aspirates that had been continuously collected year-round for the years 1987 to 2008 from infants hospitalized with acute respiratory tract illness. Phylogenetic analysis was used to assess HMPV subgroups. Results: Of the 3576 samples obtained, 202 (5.6%) tested positive for HMPV. All known HMPV subgroups (A1, A2a, A2b, B1, B2) could be identified as important respiratory tract pathogens in infants. We found that one HMPV subgroup predominated each year, and it was displaced by another subgroup every 1 to 3 years. Besides the frequent change in predominant HMPV subgroups, we observed a yearly shift in the seasonal occurrence, with a strong peak of HMPV activity in late spring-summer months every second year. Conclusion: HMPV activity is characterized by a periodic change in the predominant subgroup and it shows a stable seasonal rhythm of alternating winter and spring activity.

Biennial spring activity of human metapneumovirus in Austria.

Background: Human metapneumovirus (HMPV) is considered an important respiratory pathogen in young children. To gain insight into the seasonality and epidemiologic characteristics of HMPV infection, this study determined the frequency of HMPV infections in hospitalized infants during a 7-year period. Methods: By use of real-time reverse-transcriptase polymerase chain reaction, nasopharyngeal aspirates from 1612 infants less than 2 years of age who were hospitalized for acute respiratory tract illness were tested for the presence of HMPV. Weekly HMPV testing data were analyzed to assess the timing of HMPV activity. Season variability was estimated by comparing the onset, duration, peak, and end of outbreaks from October 2000 through October 2007. Results: Overall, 109 (6.8%) of 1612 cases of acute respiratory illness were associated with HMPV infection. Seasonal HMPV activity varied substantially from year to year, both in prevalence rates of HMPV cases and in seasonal timing of outbreaks. HMPV activity was characterized by a biennial rhythm, with spring seasons occurring every second year, and these accounted for a substantial proportion, up to 30%, of hospitalized cases of acute respiratory tract illness. Conclusions: HMPV activity varies substantially from year to year, both in the frequency and timing of illness and shows a biennial pattern of alternating winter and spring activity.

Variation of the Specificity of the Human Antibody Responses after Tick-Borne Encephalitis Virus Infection and Vaccination

ABSTRACT Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses, we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees. Postinfection and postvaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, did not play any role in the human neutralizing antibody response, which was dominated by antibodies to domains I and II. Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein, which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans. IMPORTANCE Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis, and West Nile viruses and distributed in large parts of Europe and Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses are currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human postinfection and postvaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines.