Robert S. van Binnendijk

Protective Immunity in Macaques Vaccinated with a Modified Vaccinia Virus Ankara-Based Measles Virus Vaccine in the Presence of Passively Acquired Antibodies

ABSTRACT Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.

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.

Mumps outbreak in a highly vaccinated student population, The Netherlands, 2004

In September 2004 a mumps outbreak occurred at an international hotel school in The Netherlands. We investigated this outbreak to identify risk factors for mumps. There were 105 mumps cases (overall mumps attack rate (AR) 12% (95% CI: 10-15%)). The AR for Dutch vaccinated and unvaccinated participants was 12% (95% CI: 10-15%) and 15% (95% CI: 3-42%), respectively. Independent risk factor was mumps contact. Explanations for the relatively high AR among vaccinated participants include primary vaccine failure, waning immunity and incomplete vaccine-induced immunity in the context of high mumps virus exposure in a school party and a crowded boarding school.

Identification and Characterization of Herpes Simplex Virus-Specific CD4+ T Cells in Corneas of Herpetic Stromal Keratitis Patients

Herpetic stromal keratitis (HSK) is a corneal disease initiated by a herpes simplex virus (HSV) infection with a postulated T cell-mediated immunopathology. To study the antigen specificity of cornea-infiltrating T cells in HSK patients, T cells were isolated and expanded by mitogenic stimulation from corneas of 2 patients with HSV-1-mediated HSK. A substantial number of the T cell clones (TCCs) obtained from these T cell lines were HSV-specific. All HSV-specific TCCs were of the CD3+CD4+CD8- phenotype. These TCCs responded to autologous HSV-infected corneal keratocytes, which expressed HLA class II molecules following incubation with interferon-gamma. Upon HSV-specific stimulation, all TCCs secreted interleukin-4, interleukin-5, and interferon-gamma. The data presented suggest that HSV-specific CD4+ T cells play a role in the immunopathogenesis of HSK in humans and that corneal keratocytes may act as antigen-presenting cells in this local T cell response.

Vaccine strategies to overcome maternal antibody mediated inhibition of measles vaccine.

A vaccine that is effective in the presence of maternally derived virus neutralizing antibodies and can be administered successfully at an early age, would be favoured over the presently used live attenuated measles vaccines. With the advent of new molecular and immunological techniques, several options for the development of new generation vaccines, fulfilling these criteria, have arisen. We have recently evaluated the efficacy of recombinant vaccinia virus- and iscom-based candidate vaccines, presenting the F and H proteins of measles virus, in macaques with passively transferred virus neutralizing macaque antibodies. The data indicate that the further exploration of the potential of iscom based measles vaccines should be encouraged.

Assessment of serological evidence for mumps virus infection in vaccinated children

It is estimated that at least one-third of mumps virus infections in non-vaccinated individuals are asymptomatic. Little information is available whether this proportion is the same among those vaccinated. We validated a commercial oral fluid mumps IgG-specific Enzyme Immunoassays (EIA) with vaccinated control groups to identify symptomatic and asymptomatic mumps virus infections in vaccinated individuals during a mumps outbreak in The Netherlands. A vaccinated control group was required to define a new cutoff value for the assay, because of the presence of low but significant levels of IgG antibodies in oral fluid as a result of mumps vaccination in the past. With a new cutoff, calculated using receiver operator characteristic analysis, we identified an attack rate of 7-10% compared to 2.7% based on clinical symptoms among vaccinated children. This finding has important implications when studying transmission patterns, strain virulence, as well as mumps vaccine effectiveness to protect from infection rather than disease.

Transmission of mumps virus from mumps-vaccinated individuals to close contacts

During a recent mumps epidemic in the Netherlands caused by a genotype D mumps virus strain, we investigated the potential of vaccinated people to spread mumps disease to close contacts. We compared mumps viral titers of oral fluid specimens obtained by quantitative PCR from vaccinated (n=60) and unvaccinated (n=111) mumps patients. We also investigated the occurrence of mumps infection among the household contacts of vaccinated mumps patients. We found that viral titers are higher for unvaccinated patients than for vaccinated patients during the 1st 3 days after onset of disease. While no symptomatic cases were reported among the household contacts (n=164) of vaccinated mumps patients (n=36), there were cases with serological evidence of asymptomatic infection among vaccinated household contacts (9 of 66 vaccinated siblings). For two of these siblings, the vaccinated index patient was the most probable source of infection. We conclude that, in this particular outbreak, the risk of a close contact becoming infected by vaccinated patients was small, but present.

Measles outbreak, the Netherlands, 2008.

To the Editor: From June 1 through October 16, 2008, an outbreak of 99 reported measles cases occurred in the Netherlands (1). This outbreak was the largest measles outbreak in the Netherlands since 1999–2000, when >3,200 cases, including 3 deaths, were reported (2). In the Netherlands, clinical symptoms compatible with measles in a person with laboratory-confirmed measles virus infection or an epidemiologic link to a laboratory-confirmed case are notifiable (i.e., must be reported to public health authorities). The National Measles Reference Laboratory conducts genotyping and submits sequences to the World Health Organization European Region Measles Nucleotide Surveillance database (www.hpa-bioinformatics.org.uk/Measles/Public/Web_Front/main.php). Of the 99 measles cases reported in the 2008 outbreak, 40 were laboratory confirmed and 59 were notified based on an epidemiologic link. The first case-patient in the outbreak was a 6-year-old unvaccinated resident of The Hague who had not been abroad in the month before onset of illness. The source of her infection was unknown. She attended a school based on anthroposophic principles; the school had an estimated measles-mumps-rubella (MMR) vaccination coverage of 80% (M. Monne-van Wirdum, pers. comm.). Subsequently, 52 additional cases were reported from this and from another anthroposophic school in The Hague (cluster 1; Figure A1). Two months after the first case, 22 additional cases were reported associated with an anthroposophic summer camp in the east of the Netherlands (cluster 2; Figure A1). Five additional cases had an epidemiologic link with an anthroposophic summer camp in France (cluster 3, 2 cases; Figure A1) and Switzerland (cluster 4, 3 cases; Figure A1). No known measles patients in Switzerland were linked to this cluster (J. Richard, pers. comm.). Subsequently, 12 cases were reported that were associated with 2 daycare centers in the city of Utrecht (cluster 5 and 6), both linked to an anthroposophic community. From all 6 clusters and from 2 of the 7 cases with an unknown source, indistinguishable measles viruses (genotype D8, 22 cases) were identified. Given the low prevalence of this strain in Europe (J. Kremer, pers. comm.), we concluded that virus transmission occurred between all 6 clusters. The first cluster was not epidemiologically linked to any of the recent outbreaks in anthroposophic groups in Europe (3). No case had an epidemiologic link to more than 1 cluster, suggesting the 6 cases introducing measles into these clusters were unreported. When the 7 cases with an unknown source are considered, this finding suggests that at least 13 cases were not reported (maximum reporting completeness 88%). However, transmission through patients with subclinical cases may also have played a role (4). There were no deaths. Four case-patients (4%) were admitted to hospitals. The median age was 9 years (range 8 months–48 years). Of the 98 case-patients with information on vaccination status, 91 (93%) had been unvaccinated, 6 (6%) had had 1 dose, none (0%) had had 2 doses, and 1 (1%) had had 3 doses before onset of illness. One of the 6 case-patients, vaccinated only once, had received her first MMR vaccine only 11 days before the date of onset of illness and is hence not considered a vaccine failure. Of all 99 case-patients, 91% had been eligible for >1 MMR vaccination according to the vaccination schedule in the Netherlands. Of these cases, available information for 84 case-patients indicated 48% (40 persons) were reported to be unvaccinated because of their anthroposophic beliefs, 49% (41 persons) because of a critical attitude towards vaccination, and 4% (3 persons) for other reasons. Outbreak control plans in the Netherlands focus on protecting the population by adjusting the vaccination schedule during a nationwide outbreak (5). Studies are ongoing into knowledge and attitudes toward vaccination in communities with low vaccination coverage, aiming to identify opportunities to improve coverage. The outbreak remained largely restricted to persons with philosophical objections to MMR vaccination, which suggests that there are sufficient levels of herd immunity in the general population. Remarkably, no cases were reported from the Dutch Orthodox Reformed Church community, despite the low vaccine coverage in this group. This finding suggests that orthodox reformed and anthroposophic population subgroups have little direct contact, consistent with previous observations (6). Measles vaccination was introduced in the Netherlands in 1976. The single-dose regimen was in 1987 replaced by a 2-dose regimen of MMR vaccine; the first dose at 14 months and the second at 9 years. The vaccination coverage for >1 MMR dose has been >95% from birth cohort 1986 onward (7). During 2002–2007, the incidence of measles notifications in the Netherlands was below the World Health Organization regional threshold for elimination (1/1 million population/year) (8). Nevertheless, this outbreak demonstrates the continued risk for measles transmission in the Netherlands. This suggests that indicators based merely on incidence and national vaccination coverage are of limited usefulness for certification of measles elimination. Data on measles seroprevalence and mixing patterns that will soon be available from the second national seroprevalence study will provide more insight into the dynamics of measles transmission in a population with pockets of low vaccination coverage. These data will also help assess progress toward measles elimination from the Netherlands.

Air travel as a risk factor for introduction of measles in a highly vaccinated population

Epidemiological and molecular investigation of two small measles clusters in The Netherlands in July/August 2007 revealed an association with travel by air of the index cases and nosocomial spread in the first cluster. Although these importations did not result in an outbreak among unvaccinated subjects, the observations illustrate the challenges for measles control in a country with high measles vaccination coverage (> 95%) but with pockets of low coverage.

In vitro processing and presentation of a lipidated cytotoxic T-cell epitope derived from measles virus fusion protein

Lipopeptidic formulations have been described as efficient activators of cytotoxic T lymphocytes (CTL). To better understand the pathway via which lipopeptides reach the MHC class I molecules we studied the intracellular processing and presentation of a measles virus-derived CTL epitope, to which a palmitoyl moiety was added synthetically. The palmitoyl group was conjugated to the N-terminus either directly or via a spacer sequence. The use of single or double fluorescent-labeled lipopeptides allowed the visualization of intracellular processing of these antigens using confocal microscopy. Our data indicate that the spacer composition influences internalization of the conjugate into the cell, proteasomal degradation, translocation into the ER by the transporter associated with antigen processing (TAP), and the intracellular trafficking of lipopeptides.