Rob S. van Binnendijk

Measles virus fusion protein- and hemagglutinin-transfected cell lines are a sensitive tool for the detection of specific antibodies by a FACS- measured immunofluorescence assay

A FACS-measured immunofluorescence assay was developed for the detection of antibodies directed against the hemagglutinin (H) and fusion (F) glycoproteins of measles virus (MV). Human melanoma cell lines transfected with either the MV H or F genes, which showed a high surface expression of the respective proteins in their native conformation, were used as target cells. The cells were incubated with diluted plasma samples, and stained subsequently with FITC-conjugated secondary antibodies. The FACS-measured fluorescence signals correlated directly with the amount of specific immunoglobulins over a wide concentration range. The use of different conjugates enabled the separate detection of MV-specific IgG, IgM, IgA and IgG subclasses, with relatively low backgrounds. Hemagglutinin-specific IgG, IgM and IgA fluorescence signals were shown to correlate well with MV-specific IgG ELISA titers and MV-specific IgM or IgA capture ELISA OD450-values, respectively. The polyclonal conjugates with specificity for human immunoglobulins offered sufficient cross-reactivity to detect MV-specific IgG, IgM and IgA in plasma samples of cynomolgus macaques, making this technique a useful tool for studying serological responses in vaccination and challenge experiments in non-human primate models.

Host resistance to rat cytomegalovirus (RCMV) and immune function in adult PVG rats fed herring from the contaminated Baltic Sea

Abstractu2002The immunotoxic potential of many classes of environmental contaminants has been well established in laboratory studies, with much attention being focussed on aryl hydrocarbon (Ah)-receptor binding polychlorinated biphenyl (PCB), polychlorinated dibenzo-p-dioxin (PCDD), and polychlorinated dibenzofuran (PCDF) congeners. In a semi-field study, we previously showed that harbour seals (Phoca vitulina) fed herring from the contaminated Baltic Sea had lower natural killer cell activity, T-lymphocyte functionality and delayed-type hypersensitivity responses than seals fed herring from the relatively uncontaminated Atlantic Ocean. While ethical and practical constraints preclude in-depth studies in seals, specific reagents and a wider array of immune function tests allow such studies in laboratory rats. We therefore carried out a feeding study in rats aimed at extending our observations of contaminant-induced immunosuppression in harbour seals. The same two herring batches used in the seal study were freeze-dried, supplemented and fed to female adult PVG rats for a period of 4u200912 months. Daily contaminant intakes of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents (TEQ) were estimated to be 0.3u2005ng/kg body weight and 1.6u2005ng/kg in the Atlantic and Baltic groups, respectively. At the end of the feeding experiment, no contaminant-related changes in spleen CD+4/CD+8 cellularity, natural killer cell activity, or mitogen-induced proliferative responses of thymus or spleen cells could be detected. However, total thymocyte numbers and thymus CD+4/CD+8 ratios were reduced in the Baltic group. A novel model was established to assess the specific T-cell response to rat cytomegalovirus (RCMV). When applied to the feeding study, no differences between the Atlantic and Baltic groups in the RCMV-induced proliferative T-lymphocyte responses could be detected, but virus titres in salivary glands of infected rats of the Baltic Sea group were higher. These elevated RCMV titres and changes in thymus cellularity suggest that the dietary exposure to low levels of contaminants may have been immunotoxic at a level which our immune function test could not otherwise detect. While the herring diet per se appeared to have an effect on several immune function parameters, lower plasma thyroid hormone levels in the Baltic Sea group of rats confirmed that exposure to the environmental mixture of contaminants led to adverse PHAH-related health effects.

Influenza virus-specific antibody dependent cellular cytoxicity induced by vaccination or natural infection.

Influenza viruses are responsible for substantial morbidity and mortality during seasonal epidemics. Vaccination is the most effective method to prevent infection, however due to antigenic drift of the viral surface protein hemagglutinin (HA), annual influenza virus vaccination is required. In addition to seasonal viruses, certain (avian) influenza A viruses of other subtypes, like H5N1 or H7N9, cause sporadic zoonotic infections. Therefore, the availability of game-changing novel vaccines that induce universal immune responses to a wide variety of influenza A virus subtypes is highly desirable. The quest for universal influenza vaccines has fueled the interest in broadly-reactive antibodies specific for the stalk of hemagglutinin (HA) and biological activities of antibodies other than direct virus neutralization, like antibody-dependent cellular cytotoxicity (ADCC). In the present study, we investigated the ADCC response upon influenza virus vaccination and infection in humans using a robust ADCC assay that is based on the use of recombinant HA and a continuous NK cell line that expresses FcγRIII (CD16). This assay offers advantages over existing methods, like ease to perform and possibilities to standardize. We showed that HA-specific ADCC mediating antibodies are induced by vaccination with adjuvanted trivalent seasonal and monovalent H1N1pdm09 inactivated vaccines, and by infection with H1N1pdm09 virus. In addition, the use of chimeric influenza HA with a H1 stem but antigenically irrelevant head domain derived from an avian virus allowed detection of H1-stalk-specific ADCC mediating antibodies. This assay will facilitate the assessment of ADCC mediating serum antibodies after (universal) influenza vaccination or infection and may define ADCC activity as a correlate of (cross-) protection in the future.

Mumps-specific cross-neutralization by MMR vaccine-induced antibodies predicts protection against mumps virus infection

BACKGROUNDnSimilar to other recent mumps genotype G outbreaks worldwide, most mumps patients during the recent mumps genotype G outbreaks in the Netherlands had received 2 doses of measles, mumps and rubella (MMR) vaccine during childhood. Here, we investigate the capacity of vaccine-induced antibodies to neutralize wild type mumps virus strains, including mumps virus genotype G.nnnMETHODSnIn this study, we tested 105 pre-outbreak serum samples from students who had received 2 MMR vaccine doses and who had no mumps virus infection (n=76), symptomatic mumps virus infection (n=10) or asymptomatic mumps virus infection (n=19) during the mumps outbreaks. In all samples, mumps-specific IgG concentrations were measured by multiplex immunoassay and neutralization titers were measured against the Jeryl Lynn vaccine strain and against wild type genotype G and genotype D mumps virus strains.nnnRESULTSnThe correlation between mumps-specific IgG concentrations and neutralization titers against Jeryl Lynn was poor, which suggests that IgG concentrations do not adequately represent immunological protection against mumps virus infection by antibody neutralization. Pre-outbreak neutralization titers in infected persons were significantly lower against genotype G than against the vaccine strain. Furthermore, antibody neutralization of wild type mumps virus genotype G and genotype D was significantly reduced in pre-outbreak samples from infected persons as compared with non-infected persons. No statistically significant difference was found for the vaccine strain. The sensitivity/specificity ratio was largest for neutralization of the genotype G strain as compared with the genotype D strain and the vaccine strain.nnnCONCLUSIONSnThe reduced neutralization of wild type mumps virus strains in MMR vaccinated persons prior to infection indicates that pre-outbreak mumps virus neutralization is partly strain-specific and that neutralization differs between infected and non-infected persons. Therefore, we recommend the use of wild type mumps virus neutralization assays as preferred tool for surveillance of protection against mumps virus infection.

Large measles epidemic in the Netherlands, May 2013 to March 2014: changing epidemiology

Since the early 1990s, the Netherlands has experienced several large measles epidemics, in 1992–94, 1999–2000 and in 2013–14. These outbreaks mainly affected orthodox Protestants, a geographically clustered population with overall lower measles-mumps-rubella first dose (MMR-1) vaccination coverage (60%) than the rest of the country (>u200995%). In the 2013–14 epidemic described here, which occurred between 27 May 2013 and 12 March 2014, 2,700 cases were reported. Several control measures were implemented including MMR vaccination for 6–14-month-olds and recommendations to reduce the risk in healthcare workers. The vast majority of reported cases were unvaccinated (94%, nu2009=u20092,539), mostly for religious reasons (84%, nu2009=u20092,135). The median age in the epidemic was 10 years, 4 years older than in the previous epidemic in 1999–2000. A likely explanation is that the inter-epidemic interval before the 2013–2014 epidemic was longer than the interval before the 1999–2000 epidemic. The size of the unvaccinated orthodox Protestant community is insufficient to allow endemic transmission of measles in the Netherlands. However, large epidemics are expected in the future, which is likely to interfere with measles elimination in the Netherlands and elsewhere.

Comparison of norovirus genogroup I, II and IV seroprevalence among children in the Netherlands, 1963, 1983 and 2006

Noroviruses are a major cause of acute gastroenteritis worldwide and are a genetically diverse group of viruses. Since 2002, an increasing number of norovirus outbreaks have been reported globally, but it is not clear whether this increase has been caused by a higher awareness or reflects the emergence of new genogroup II genotype 4 (GII.4) variants. The hypothesis that norovirus prevalence has increased post-2002 and is related to the emergence of GII.4 is tested in this study. Sera collected from children aged <5 years of three Dutch cross-sectional population based cohorts in 1963, 1983 and 2006/2007 (n=143, n=130 and n=376, respectively) were tested for specific serum IgG by protein array using antigens to GII.4 and a range of other antigens representing norovirus GI, GII and GIV genotypes. The protein array was validated by paired sera of norovirus infected patients and supernatants of B-cell cultures with single epitope specificity. Evidence for norovirus infection was found to be common among Dutch children in each cohort, but the prevalence towards different genotypes changed over time. At the genogroup level, GI seroprevalence decreased significantly between 1963 and 2006/2007, while a significant increase of GII and, in particular, specific antibodies of the genotype GII.4 was detected in the 2006/2007 cohort. There were no children with only GII.4 antibodies in the 1963 cohort. This study shows that the high GII.4 norovirus incidence in very young children is a recent phenomenon. These findings are of importance for vaccine development and trials that are currently focusing mostly on GII.4 viruses.

Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission

Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering.

Polio and Measles Down the Drain: Environmental Enterovirus Surveillance in the Netherlands, 2005 to 2015.

ABSTRACT Polioviruses (PVs) are members of the genus Enterovirus. In the Netherlands, the exclusion of PV circulation is based on clinical enterovirus (EV) surveillance (CEVS) of EV-positive cases and routine environmental EV surveillance (EEVS) conducted on sewage samples collected in the region of the Netherlands where vaccination coverage is low due to religious reasons. We compared the EEVS data to those of the CEVS to gain insight into the relevance of EEVS for poliovirus and nonpolio enterovirus surveillance. Following the polio outbreak in Syria, EEVS was performed at the primary refugee center in Ter Apel in the Netherlands, and data were compared to those of CEVS and EEVS. Furthermore, we assessed the feasibility of poliovirus detection by EEVS using measles virus detection in sewage during a measles outbreak as a proxy. Two Sabin-like PVs were found in routine EEVS, 11 Sabin-like PVs were detected in the CEVS, and one Sabin-like PV was found in the Ter Apel sewage. We observed significant differences between the three programs regarding which EVs were found. In 6 sewage samples collected during the measles outbreak in 2013, measles virus RNA was detected in regions where measles cases were identified. In conclusion, we detected PVs, nonpolio EVs, and measles virus in sewage and showed that environmental surveillance is useful for poliovirus detection in the Netherlands, where live oral poliovirus vaccine is not used and communities with lower vaccination coverage exist. EEVS led to the detection of EV types not seen in the CEVS, showing that EEVS is complementary to CEVS. IMPORTANCE We show that environmental enterovirus surveillance complements clinical enterovirus surveillance for poliovirus detection, or exclusion, and for nonpolio enterovirus surveillance. Even in the presence of adequate surveillance, only a very limited number of Sabin-like poliovirus strains were detected in a 10-year period, and no signs of transmission of oral polio vaccine (OPV) strains were found in a country using exclusively inactivated polio vaccine (IPV). Measles viruses can be detected during an outbreak in sewage samples collected and concentrated following procedures used for environmental enterovirus surveillance.

Effectiveness of Early Measles, Mumps, and Rubella Vaccination Among 6–14-Month-Old Infants During an Epidemic in the Netherlands: An Observational Cohort Study

BackgroundnRoutinely, the first measles, mumps, and rubella (MMR) vaccine dose is given at 14 months of age in the Netherlands. However, during a measles epidemic in 2013-2014, MMR vaccination was also offered to 6-14-month-olds in municipalities with <90% MMR vaccination coverage. We studied the effectiveness of the early MMR vaccination schedule.nnnMethodsnParents of all infants targeted for early MMR vaccination were asked to participate. When parent(s) suspected measles, their infants saliva was tested for measles-specific antibodies. The vaccine effectiveness (VE) against laboratory-confirmed and self-reported measles was estimated using Cox regression, with VE calculated as 1 minus the hazard ratio.nnnResultsnThree vaccinated and 10 unvaccinated laboratory-confirmed cases occurred over observation times of 106631 and 23769 days, respectively. The unadjusted VE against laboratory-confirmed measles was 94% (95% confidence interval [CI], 79%-98%). After adjustment for religion and siblings vaccination status, the VE decreased to 71% (-72%-95%). For self-reported measles, the unadjusted and adjusted VE was 67% (40%-82%) and 43% (-12%-71%), respectively.nnnConclusionsnInfants vaccinated between 6 and 14 months of age had a lower risk of measles than unvaccinated infants. However, part of the effect was caused by herd immunity, since vaccinated infants were more likely to be surrounded by other vaccinated individuals.

Mumps virus pathogenesis: Insights and knowledge gaps.

ABSTRACT The recent mumps outbreaks among MMR vaccinated persons have raised questions about the biological mechanisms related to mumps symptoms and complications in the background of waning immunity. Contrary to other paramyxoviruses, the understanding of mumps virus pathogenesis is limited, and further in-depth clinical studies are required to provide answers to important research questions.