Tjeerd G. Kimman

Experimental quantification of vaccine-induced reduction in virus transmission

Although reduction in transmission of an agent in the host population is an important goal of many vaccinations, suitable experimental methods to measure transmission have been lacking. Therefore, we designed and tested an animal experiment to quantify transmission among vaccinated and unvaccinated animals. We used Aujeszkys disease virus (ADV) in pigs, because a serological test was available to detect infection in vaccinated pigs and because vaccination against ADV will be used in an attempt to eliminate ADV from the Netherlands. Our experiments showed that vaccinating twice with vaccine 783 significantly reduces ADV transmission. In unvaccinated groups, the estimated maximum number of secondary cases per infectious individual, i.e. the basic reproduction ratio R0, was 10.0. In contrast, the reproduction ratio for the vaccinated groups R, i.e. the average number of secondary cases per infectious individual in a totally vaccinated population, was 0.5. These results show that it is possible to measure transmission experimentally. Therefore, such measurements should be obtained for all vaccines that are intended to eliminate agents causing animal diseases, either on a single farm or in a whole country.

Association of Severe Respiratory Syncytial Virus Bronchiolitis with Interleukin-4 and Interleukin-4 Receptor α Polymorphisms

The association of variants of genes encoding interleukin (IL)-4 and the IL-4 receptor alpha chain (IL-4Ralpha) with respiratory syncytial virus (RSV) bronchiolitis was examined in hospitalized infants. Polymorphisms in IL-4 (C-590T) and IL-4Ralpha (I50V and Q551R) were genotyped by restriction fragment-length polymorphism analysis. Control subjects included parents of the hospitalized children (for the transmission/disequilibrium test), and a random population sample (for the case-control study). Results were also analyzed in a combination of these 2 tests, using Fishers method. The IL-4 590T allele was found more frequently among children hospitalized with RSV than expected in the case-control (odds ratio [OR], 1.43; P=.04) and combination (OR, 1.41; P=.02) tests. Among children who were >6 months old when they were hospitalized, compared with the control group or with the <6 months old who were hospitalized for RSV infection, higher frequencies of both the IL-4 590T allele and the IL-4Ralpha R551 allele were found. These results indicate that gain-of-function variants of T helper type 2 cytokine genes may play a role in increasing the severity of RSV disease, which appears more pronounced after the first half-year of life.

Contribution of single genes within the unique short region of Aujeszky's disease virus (suid herpesvirus type 1) to virulence, pathogenesis and immunogenicity

Pigs (3 and 10 weeks old) were infected intranasally with Aujeszkys disease virus (ADV) mutants that functionally lacked one of the non-essential genes in the unique short region of the genome (except the gene encoding the 11K protein). Virus excretion in oropharyngeal fluid and disease symptoms were monitored. Some pigs were killed to study pathogenesis, whereas others were challenged with virulent ADV 8 weeks after the primary infection. Mutants lacking protein kinase, or glycoproteins gp63 or gI showed reduced virulence, but mutants lacking gX or the 28K protein showed normal virulence. Glycoprotein gI appears to affect the tissue tropism of ADV in pigs, presumably by facilitating the spread of the virus through the central nervous system. In this study, there was no correlation between virulence and virus multiplication in either cultured cells or in the oropharynx in vivo. All mutants induced neutralizing antibody and complete or partial protection against challenge infection. Complete protection was obtained by inoculation with the gI and gX mutants, whereas incomplete protection was obtained using gp63 and protein kinase mutants. Complete clinical and virological protection was associated with the absence of secondary antibody responses in the serum.

Influence of Promoter Variants of Interleukin-10, Interleukin-9, and Tumor Necrosis Factor–α Genes on Respiratory Syncytial Virus Bronchiolitis

Previously, we reported genetic associations between severe respiratory syncytial virus (RSV) bronchiolitis in infants and polymorphisms in the interleukin (IL)-4 and IL-4 receptor alpha (IL-4Ralpha) genes, providing evidence for involvement of T helper type 2 cytokines in the pathogenesis of RSV bronchiolitis. We expanded our studies to polymorphisms in genes encoding IL-9, IL-10, and tumor necrosis factor (TNF)-alpha, using both a transmission/disequilibrium test and a case-control approach. Children homozygous for the IL-10 -592C or -592A allele had a higher risk of hospitalization for RSV bronchiolitis than did heterozygous carriers (odds ratio [OR], 1.73 vs. 2.55; 95% confidence interval [CI], 1.13-2.66 vs. 1.21-5.39). In children hospitalized at < or =6 months of age, a significant association between RSV bronchiolitis and the IL-10 -592C allele was found (OR, 1.61; 95% CI, 1.10-2.35). No significant associations of TNF-alpha and IL-9 polymorphisms with RSV bronchiolitis were observed. We also explored the interactions between different polymorphisms and found an interaction between the IL-4Ralpha Q551R and IL-10 C-592A polymorphisms.

Preclinical and clinical safety studies on DNA vaccines.

DNA vaccines are based on the transfer of genetic material, encoding an antigen, to the cells of the vaccine recipient. Despite high expectations of DNA vaccines as a result of promising preclinical data their clinical utility remains unproven. However, much data is gathered in preclinical and clinical studies about the safety of DNA vaccines. Here we review current knowledge about the safety of DNA vaccines. Safety concerns of DNA vaccines relate to genetic, immunologic, toxic, and environmental effects. In this review we provide an overview of findings related to the safety of DNA vaccines, obtained so far. We conclude that the potential risks of DNA vaccines are minimal. However, their safety issues may differ case-by-case, and they should be treated accordingly.

Both immunisation with a formalin-inactivated respiratory syncytial virus (RSV) vaccine and a mock antigen vaccine induce severe lung pathology and a Th2 cytokine profile in RSV-challenged mice.

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children. Immunopathology may play a role in RSV-induced disease and a severe RSV infection may also be associated with an increased risk of developing asthma. Vaccination with formalin-inactivated RSV (FI-RSV) prior to infection resulted both in human and in the mouse model in extensive lung pathology. In the mouse model, it has been shown that this aggravation of disease was associated with a shift in the balance between Th1 and Th2 cytokines towards a Th2-type response. The aim of the present study was to characterise the immunological and inflammatory responses in BALB/c mice upon RSV infection with or without prior vaccination with aluminium-adjuvanted FI-RSV or control antigens (FI-Mock). As previously reported by others, we also observed that a primary RSV infection in BALB/c mice resulted in a predominant Th1-type cytokine response, which was associated with slight bronchiolitis and alveolitis. FI-RSV vaccination prior to RSV challenge prevented virus replication and was associated with an aggravation of pulmonary histopathology and a shift towards a Th2-type response. Vaccination with FI-Mock did not prevent RSV replication in the lung but resulted in an even more pronounced Th2 response after infection while these mice were not sensitised to specific viral antigens. Thus, viral replication in a Th2 responding animal (induced by aluminium-adjuvanted mock vaccine) appears to boost the Th2 response upon RSV infection.

Combining the transmission disequilibrium test and case-control methodology using generalized logistic regression.

To study the role of genetic factors in the etiology, susceptibility, or severity of disease, several methods are available. In a transmission disequilibrium test, genotypes of cases are compared to those of their parents to explore whether a specific allele, or marker, at a locus of interest appears to be transmitted in excess of what is expected on the basis of Mendelian inheritance. Such apparent excess transmission indicates that cases are being selected for that allele, thereby providing evidence that this allele is a risk factor for disease. In case–control studies, genotypes of cases are compared to those of controls from the same population to identify whether a specific allele is associated with disease. If so, either the allele at this locus or one in linkage disequilibrium with it may be causally related to the etiology of the disease. Here, we discuss the problem of combining a transmission disequilibrium test and a case–control comparison, in order to integrate all available information, and thereby increase statistical power. As the same cases are used in both approaches, the two results are not independent. However, parents of cases can be independently compared to controls. Both the issue of testing for a genetic effect and the estimation of relative risks under the multiplicative model using generalized logistic regression are discussed.

Host Transcription Profiles upon Primary Respiratory Syncytial Virus Infection

ABSTRACT Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract infection in children. Severe RSV disease is related to an inappropriate immune response to RSV resulting in enhanced lung pathology which is influenced by host genetic factors. To gain insight into the early pathways of the pathogenesis of and immune response to RSV infection, we determined the transcription profiles of lungs and lymph nodes on days 1 and 3 after infection of mice. Primary RSV infection resulted in a rapid but transient innate, proinflammatory response, as exemplified by the induction of a large number of type I interferon-regulated genes and chemokine genes, genes involved in inflammation, and genes involved in antigen processing. Interestingly, this response is much stronger on day 1 than on day 3 after infection, indicating that the strong transcriptional response in the lung precedes the peak of viral replication. Surprisingly, the set of down-regulated genes was small and none of these genes displayed strong down-regulation. Responses in the lung-draining lymph nodes were much less prominent than lung responses and are suggestive of NK cell activation. Our data indicate that at time points prior to the peak of viral replication and influx of inflammatory cells, the local lung response, measured at the transcriptional level, has already dampened down. The processes and pathways induced shortly after RSV infection can now be used for the selection of candidate genes for human genetic studies of children with severe RSV infection.

Evaluation of Serological and Virological Tests in the Diagnosis of Clinical and Subclinical Measles Virus Infections during an Outbreak of Measles in The Netherlands

We evaluated different approaches for diagnosing measles virus (MV) infection in unvaccinated children and in healthy contact persons (n=194) during a measles epidemic in The Netherlands. MV RNA was detected by reverse-transcriptase polymerase chain reaction in throat-swab specimens from 93% of the patients with clinical symptoms. MV RNA was detected from 5 days before until 12 days after the onset of symptoms. Most patients (88%) also secreted MV RNA in their urine until 5 weeks after the onset of symptoms. Oral fluid proved to be the most practical specimen for the simultaneous detection of MV-specific IgM antibody and viral RNA, which, together, confirmed 93% of measles cases. Viral RNA was also detected in oropharyngeal specimens from 3 healthy contact persons with serological proof of MV infection. The results of this study emphasize the feasibility of combined detection of viral RNA and MV-specific IgM antibodies in oropharyngeal specimens for the diagnosis of clinical and subclinical MV infection.

Genetic susceptibility to respiratory syncytial virus bronchiolitis in preterm children is associated with airway remodeling genes and innate immune genes.

Prematurity is a risk factor for severe respiratory syncytial virus bronchiolitis. We show that genetic factors in innate immune genes (IFNA13, IFNAR2, STAT2, IL27, NFKBIA, C3, IL1RN, TLR5), in innate and adaptive immunity (IFNG), and in airway remodeling genes (ADAM33 and TGFBR1), affect disease susceptibility to a different extent in preterm children, born with underdeveloped lungs, than in term children.