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Vaccination has reduced the disease burden of vaccine-preventable diseases. However, the extent to which seasonal cycles of immunity could influence vaccine-induced immunity is not well understood. A national cross-sectional serosurveillance study performed in the Netherlands (Pienter-2) yielded data to investigate whether season of vaccination was associated with antibody responses induced by DT-IPV (diphtheria, tetanus and poliomyelitis), MMR (measles, mumps and rubella) and meningococcus C (MenC) vaccines in children. In total, 434 children met the inclusion criteria to study DT-IPV immunity, 811 for MMR and 311 for MenC. Differences in log(antibody levels) by season of vaccination were investigated with linear multivariable regression analyses. Seroconversion rates varied according to season of vaccination for rubella (90% of autumn-vaccinated childrenvs.99% of winter-vaccinated had concentrations above cut-off levels). Summer-vaccinated boys showed a slower decline of tetanus antibodies (6% per month), in comparison with winter-vaccinated boys. In conclusion, season of vaccination showed little association with immunological protection. However, a number of associations were seen with aP-value of about 0.03; and adding data from a just-completed nationwide serological study might add more power to the current study. Further immunological and longitudinal investigations could help understand the mechanisms of seasonal influence in vaccine-induced responses.

The most important neutralizing and protective antibodies against Measles virus (MeV) are directed against the hemagglutinin protein (MeV-H). To define the MeV binding domains recognized by human antibodies a set of 10 non-redundant MeV-H-specific monoclonal antibodies (mabs) was used to block their binding in a competition ELISA. Sera from both naturally infected and vaccinated individuals showed similar competition patterns. Two distinct domains were identified as the main target of human antibodies. One domain corresponded to the region of the previously described hemagglutinin noose epitope (HNE, aa 380-400) [35], which is recognized by hemagglutination-inhibiting, neutralizing and protective mabs. The second region is defined by a mab with strong neutralizing but weak hemagglutination-inhibiting activity. Mabs with a strong neutralizing capacity with respect to wild-type viruses seemed to displace more human antibodies than those with a weaker neutralizing activity. Human antibodies seem to react more weakly with the hemagglutinin regions that bind the CD46 and the fusion protein and more strongly with the putative CD150 binding site and the top loops of beta-sheet 2 and 3 of the hemagglutinin.

The immunity to vaccine-preventable diseases included in the Dutch immunisation programme in the general population and among orthodox reformed individuals who refuse vaccination was assessed. The programme induces good protection. However, a large proportion of adults lacks diphtheria and tetanus immunity. Measles, mumps and rubella seroprevalence was somewhat lower among vaccinated compared to unvaccinated cohorts. The prevalence of HibPS antibodies declined during 2.5 years after the fourth vaccination. However, protection occurs also by memory immunity. Herd immunity is sufficient among the general population, but not among orthodox reformed individuals. Immunosurveillance is an efficient way to evaluate the effects of immunisation programmes and identify risk groups for infection.

We assessed antibodies to the capsular polysaccharide of Haemophilus influenzae type b (HibPS) in the Dutch population a few years after a mass vaccination against H. influenzae (Hib) was begun. We observed sharp declines in the geometric mean titer (GMT) and the prevalence of HibPS antibodies at levels ofor =0.15 microg/mL in children who had received 4 doses of vaccine: from 8.65 microg/mL (prevalence, 99.4%) after 0-2 months to 0.8 microg/mL (prevalence, 83.3%) after 27-29 months. In adult groups, both the prevalence of HibPS antibodies and the GMT declined significantly with increasing age but remained high (prevalence,or =83.7%; GMT, 0.73or = microg/mL). We conclude that the overall immunity in the Dutch population seems satisfactory. We draw our conclusions from the current serosurveillance study and from the sharp decline in invasive Hib disease noted after the introduction of vaccination. The key questions for the future are (1) whether Hib and cross-reacting organisms will circulate sufficiently to provide natural reexposure, and (2) how long memory immunity will persist after vaccination without reexposure.

We assessed the tetanus immunity of the general Dutch population and of religious groups refusing vaccination by means of population-based study to evaluate the effect of tetanus vaccination. More than 95% of those born after the introduction of routine vaccination had tetanus antitoxin levels above the minimum protective level. After the sixth vaccination, a fall in tetanus antitoxin levels occurred. Nevertheless, immunisation in accordance with the routine programme most likely induces protection for much longer than two decades. Not only many members of religious groups who refuse vaccination, but also many adults born before the introduction of vaccination lack tetanus immunity. These cohorts might benefit most from (re)vaccination.

The haemagglutinin protein (H) of measles virus (MV) binds to susceptible cells and collaborates with the fusion protein (F) to mediate fusion of the virus with the cell membrane. Binding and fusion activity of the virus can be monitored by haemagglutination and haemolysis, respectively, of monkey erythrocytes. Most monoclonal antibodies (MAbs) with haemolysis inhibiting activity (HLI+) are either MV-F specific and do not inhibit haemagglutination (HI-), or they bind to MV-H and are HI+ by interfering with virus binding. We describe here a small panel of H-specific MAbs (BH47, BH59, BH103, BH129) which bind to a new linear neutralizing epitope, H244-250 (SELSQLS; NE domain), and which prevent virus-cell fusion (HLI+) but not virus binding (HI-). These antibodies also protect against MV encephalitis in an animal model. They do not compete with an HLI+/HI+ antibody (BH216) which binds to the haemagglutinin noose epitope (HNE). The antibodies described here and the HNE-specific antibodies are functionally distinct and define two topographically non-overlapping interfaces, supposedly with a bias towards the host cell MV-receptor and the fusion protein respectively. The proximity of the CD46 downregulating amino acid Arg-243 may suggest a functional link between the domain described here and the CD46 binding domain. This new protective linear site is also of potential interest for the design of a subunit-based vaccine.

A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA.

In 2006/2007 a large serum bank was established by means of a cross-sectional population-based study. This serum bank will be used to evaluate the Dutch national immunisation programme (NIP) by serosurveillance and additional immunological and epidemiological research. In this paper we describe the design of this population-based cross-sectional serosurvey and report the participation rates as well as general characteristics of the study population. A similar serum bank was collected in 1995/1996. Dutch inhabitants (aged 0-79 years, men and women) were invited from 40 municipalities throughout the country and also from eight additional municipalities known with low vaccination coverage (LVC). An oversampling of the migrant population was performed. Blood samples were obtained from all participants accompanied with extensive information on demographic and epidemiological data, such as vaccination history, risk factors and travelling. In addition, sociodemographic data are available from individuals who declined to participate (non-response survey). Overall 33% of all invitees were included in this study. The serum bank comprises 6386 sera in the nationwide sample including the extra sample of immigrants (n=646) and 1518 sera from the LVC municipalities. The sera will be analysed for antibodies against all NI P antigens but will also be used for other infectious diseases research. Results of this second serosurveillance study will contribute to the discussion whether it is needed to reconsider the schedule and/or the vaccine components of the current National Immunisation Programme.

The isolated lenticular plasma membrane-cytoskeleton complex, when analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis, shows reproducibly a significant amount of crystallins, mainly alpha-crystallin. Evidence is provided that purified plasma membranes from the bovine lens also associate selectively with a limited number of newly synthesized polypeptides on cell-free translation of calf lens polyribosomes and addition of the membranes to the incubation medium. This capability is retained by purified lens membrane junctions. The polypeptides that are selected comprise alpha-crystallin chains (in particular alpha A2-crystallin), actin, vimentin and beta B1a-crystallin. Sequence analysis revealed that the latter has in its N-terminal extension a characteristic Pro-Ala track. The designation 'PAPA-arm' is proposed for this N-terminal region, comprising the alternating Pro-Ala sequence, that has previously also been found in rabbit myosin and might be responsible for anchoring beta B1a-crystallin to lens membranes.

All infants in the Netherlands, which are born after March 2006, receive additional vaccinations at the age of 2, 3, 4 and 11 months to protect them against pneumococcal infections. During the same visit to a consultation bureau, the children also receive a combination vaccine against diphtheria, pertussis, tetanus, poliomyelitis and Haemophilus influenzae (DTPa-IPV-Hib). Children of which at least one parent was born in a country where hepatitis B occurs relatively often are also vaccinated in the Netherlands against hepatitis B. This currently pertains to about 15% of all newborns. These children now receive a new combination vaccine in which a hepatitis B component has been added to the DTPa-IPV-Hib components. They will receive this combination vaccine 4 times. This combination vaccine is given during the same visit as the pneumococcal vaccination. Although pneumococcal vaccination may have a somewhat negative effect on the immune response to hepatitis B, it is expected that the new 4-fold vaccination schedule will induce good and long-lasting protection against hepatitis B in the vast majority of the children. About 700 children are born out of mothers infected with hepatitis B each year in the Netherlands. In the new vaccination schedule, they now receive 5 active vaccinations against hepatitis B and are examined serologically on an individual basis in order to detect breakthrough infections. This will also generate greater insight into the efficacy of the different vaccination schemes and intervention programmes to prevent vertical transmission of the virus.

The immunity to vaccine-preventable diseases included in the Dutch immunisation programme in the general population and among orthodox reformed individuals who refuse vaccination was assessed. The programme induces good protection. However, a large proportion of adults lacks diphtheria and tetanus immunity. Measles, mumps and rubella seroprevalence was somewhat lower among vaccinated compared to unvaccinated cohorts. The prevalence of HibPS antibodies declined during 2.5 years after the fourth vaccination. However, protection occurs also by memory immunity. Herd immunity is sufficient among the general population, but not among orthodox reformed individuals. Immunosurveillance is an efficient way to evaluate the effects of immunisation programmes and identify risk groups for infection.

A two-dose mass vaccination programme with a combined vaccine against measles, mumps and rubella (MMR) was adopted in the Netherlands in 1987, replacing the selective schoolgirl vaccination strategy introduced in 1974. To obtain insight into the effect of mass vaccination and the population's immunity, the antibody levels against rubella were studied in the general Dutch population and in religious groups refusing vaccination. In the national sample, we observed a high prevalence (96·5%) for rubella antibodies in vaccinated cohorts as well as in the older unvaccinated cohorts. No indications of rapidly waning immunity after vaccination were found. There are indications of low virus circulation in the last few years. The very high seroprevalence in women at childbearing age is consistent with the few reported cases of congenital rubella syndrome (CRS) at present. However, individuals in the age group of 1–9 years who are not vaccinated for religious or other reasons have a considerably lower seroprevalence and thus there is a potential risk of a CRS outbreak in the future.

The amino acid sequence of the N-terminal region of the two basic bovine beta-crystallin B1 chains has been analyzed. The results reveal that beta B1b is derived in vivo from the primary gene product beta B1a by removal of a short N-terminal sequence. It appears that the beta B1 chains have the same domain structure as observed in other beta- and gamma-crystallin chains. They have, however, a very long N-terminal extension in comparison with other beta-chains. This extension is mainly composed of a remarkable Pro- and Ala-rich sequence, which suggests an interaction of these structural proteins with the cytoskeleton and/or the plasma membranes of the lens cells.

A Ca2+-dependent transglutaminase (EC 2.3.2.13) has been demonstrated in the eye lenses of several mammalian species [Lorand, L., Hsu, L. K. M., Siefring, G. E., Jr., & Rafferty, N. S. (1981) Proc. Natl. Acad. Sci. USA 78, 1356-1360]. Using [3H]methylamine as a convenient probe for transglutaminase activity, we have explored the action of this enzyme in the bovine eye lens. We could characterize the glutamine residues acting as acyl-donor sites in three beta-crystallin chains, which are the only substrates for lens transglutaminase among the various lens-specific structural proteins, the crystallins. A single glutamine was found to bind [3H]methylamine in each of these three chains: glutamine -9 in beta Bp (beta B2), glutamine -21 in beta B3, and glutamine -23 or -24 in beta A3. The four glutamines are all located in the NH2-terminal regions, which presumably extend from the compact two-domain structure of the beta-crystallin chains. It was, moreover, established that several components of the lens cytoskeleton are substrates for transglutaminase.

Large quantities of calf gamma-crystallin can be prepared by a single and rapid salting-out procedure. The final product is indistinguishable from the gamma-crystallin fraction obtained after gel filtration of a 15000 g lens protein supernatant over Sephadex G 200. Further fractionation is achieved by the mild procedure of chromatofocusing yielding six to eight subfractions. The latter have been characterized by polyacrylamide gel electrophoresis (PAGE) in 6 M-urea, SDS-gel electrophoresis, partial digestion with Staphylococcus aureus protease and amino acid analysis.

Our knowledge of the structural and functional effects of post-translational modifications of proteins is still fragmentary. It is in general implicitly assumed that such modifications must have important biological roles, in the regulation of enzyme activities, protein interactions or turnover. Although such functions seem plausible in many instances, definite proof is often difficult to provide, and in fact it cannot be excluded that certain modifications are functionally unimportant and just occur as innoxious by-products of the intracellular metabolism. For a better understanding of the meaning of post-translational modifications it is important to analyze and compare their occurrence and features in different proteins and cell types. This contribution reports some recent findings on post-translational modifications of eye lens proteins.

The bovine β-crystallin B p chain is organized into two very similar domains, with short extensions at both N - and C-termini, and two alternative models for the βB p dimer have been proposed (Wistow, G., Slingsby, C., Blundell, T., Driessen, H.P.C., De Jong, W.W. And Bloemendal, H. (1981) FEBS Lett. 133, 9–16). By limited proteolysis the C-terminal arms can be cleaved off rapidly from the βB p dimer, while the N-terminal arms are more difficult to remove. Trypsin divides the βB p chain into two fragments which approximately correspond to the two structural domains. Dissociation and reassociation of the different products of limited proteolysis indicated that: the C-terminal arm extends freely from the surface and is not involved in subunit-contact; at least one N-terminal arms seems required for dimer formation; the N-terminal domains have a greater tendency to associate than the C-terminal domains and, when mixed, the purified domains reassociate partially to a M r 50 000 structure like native βB p . These findings support the more extended dimer model of βB p .