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Chimeric molecules expressing multiple copies of the loop-forming hemagglutinin noose epitope (designated as "L"; aa386-400), a protective B cell epitope of the measles virus were generated by recombinant technology. The recombinant polyepitope [L4T4]2 combining two sets of four repeats of the L epitope and with two sets of four repeats of the human promiscuous T cell epitope of tetanus toxoid ("T", tt830-844) was produced in transgenic carrot plants. After intraperitoneal immunization of mice with plant membrane extract, sera neutralized all wild-type viruses. In a modified plaque reduction neutralization assay based on CD150-transfected Vero cells anti-[L4T4]2 sera neutralized all field isolates, irrespective of mutations in the L epitope. Even viruses with a mutation in the contact residues of a neutralizing L-specific monoclonal antibody or two mutations in other positions of the epitope were equally sensitive to neutralization. These results suggest that the multiple copies of the L epitope fold into different conformations that induce a repertoire of B cells diverse enough to overcome the genetic diversity of field viruses.

Maternal and cord blood collected from 33 Nigerian mother-child pairs were tested for measles-sepcific IgG. All 33 had protective measles antibodies at the time of delivery with a positive correlation of r = 0.87. Determination of the rate of waning of these antibodies revealed that 58 per cent of these children had lost the protective maternal antibody by the age of 4 months and only 3 per cent of the children had enough antibody to protect them between the ages of 6-9 months. Fifty-five colostrum samples from the same mothers and 347 breastmilk samples collected at various periods of breastfeeding also showed that anti-measles IgA had dropped below the protective cut-off within the first 2 weeks of birth. It is evident that the Nigerian child is born with solid anti-measles antibody but the rate of waning has left a large number unprotected before the first dose of the vaccine. There is an urgent need to review the measles vaccination programme in Nigeria to protect these susceptible infants.

The current live-attenuated measles vaccine leaves many children unprotected until they reach the recommended age of vaccination. We have previously shown that the short peptide corresponding to the hemagglutinin noose epitope (HNE) of the measles virus (MV) hemagglutinin protein induced virus-neutralizing antibodies even in the presence of protective levels of anti-whole virus-specific antibodies. Here we investigate the immunogenicity of HNE peptide-conjugates of diphtheria or tetanus toxoid in mice after active and passive priming with antibodies against the peptide, toxoids and conjugates. Both conjugates induced high titers of peptide antibodies which crossreacted with the virus and protected against a lethal intracranial challenge with a rodent-adapted measles virus, even after active priming with homologous or heterologous toxoid or conjugate. Peptide-specific epitopic suppression was stronger after passive priming with carrier or conjugate antibodies, but diphtheria toxoid as a carrier was less susceptible to suppression than tetanus toxoid and suppression was overcome by an additional boost. Furthermore, prior immunization with peptide-conjugate did not interfere with the development of a complete response to a subsequent injection of MV, suggesting that the benefits of a follow-up vaccination with the current live-attenuated vaccine would not be lost. These results underline the potential of these peptide-based conjugates as vaccine candidates for use in early infancy to close the window of susceptibility before the live-attenuated vaccine can be administered.

Sub-Saharan Africa suffers from an excessively high endemicity of hepatitis B virus (HBV), but little is known about the prevalent genotypes. In this study, we investigated the PreS1/PreS2/S genes of 127 viruses obtained from 12 locations in Mali, Burkina Faso, Togo, Benin, Nigeria, Cameroon, and the Democratic Republic of Congo. Except for those obtained from the Cameroon HIV cohort (18/22 HBV genotype A), 96 of 105 sequences belonged to HBV genotype E (HBV/E), and viral DNA was very similar (1.67% diversity) throughout this vast HBV/E crescent, which spans 6000 km across Africa. The low diversity suggests that HBV/E may have a short evolutionary history. Considering a typical mutation rate of DNA viruses, it would take only 200 years for the strain diversity of HBV/E viruses to develop from a single introductory event. The relatively recent introduction of HBV/E into humans would also explain its conspicuous absence in the Americas, despite the forced immigration of slaves from west Africa, until the early 19th century. Infection during infancy is mostly associated with chronic carrier status, and this combination can account for the explosive spread of virtually identical viruses within a community, but whether other routes of long-range transmissions must be considered becomes an important question.

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the "gold standard," the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization.

Despite vaccination, measles remains a burden in both developed and developing countries and complications may necessitate an efficient therapy. Measles virus (MEV) is susceptible to ribavirin (RBV), but the use of this drug is limited by its toxicity. Cyclodextrins (CDs) can form complexes with numerous molecules, improving their bioavailability and their biological properties. We have evaluated in vitro the antiviral effects of complexes of RBV with alpha-, beta- or gamma-CD against two clade A laboratory strains of MEV (Edmonston and CAM/RB) grown on Vero cells. Complexation of RBV with alpha-CD or beta-CD lead to a five-fold or a two-fold decrease in the 50% inhibitory concentration, respectively, against both MEV strains. In contrast, gamma-CD complexation showed no modification.

Fifty-eight outbreaks of Infectious bursal disease virus (IBDV) were observed in vaccinated chicken flocks in four Southwestern states of Nigeria between 1995 and 2000. Bursa samples from 40 flocks were found virus-positive in VP2-specific nested RT-PCR. Sequences of the hypervariable region of VP2 were compared to reference strains of the different IBDV variants including also 1988 isolates from Nigeria. Sequence analysis revealed that all 40 Nigerian isolates belonged to the very virulent (vv) variant. The maximum sequence diversity of 5.7% was higher than in all other vvIBDV sequences listed in Genbank (3.6%). Two clusters within Nigerian isolates are unique to this region. Serotype 1 IBDV was also detected in four symptomatic turkey flocks. The turkey isolates were found within 2 of the 3 VV-clusters of chicken isolates. Full length sequence of a turkey isolate (NIE009t) confirmed its close relation to vvIBDV strain D6948NET for both segment A (1.4% sequence diversity) and segment B (2.1%). Thus, turkeys should be considered susceptible to vvIBDV infection. The unusually high sequence diversity of vvIBDV may be an indication of a West-African origin of this virus, from where it spread to other continents.

Sera samples from seven poultry farms in southwest Nigeria consisting of 7 broiler, 10 pullet, 1 layer, 1 cockerel, and 1 broiler breeder flocks were tested for the presence of chicken infectious anemia virus (CIAV) antibodies using a commercial enzyme-linked immunosorbent assay kit. Eleven of the 20 flocks (55%) and six out of seven (86%) farms were positive for CIAV antibodies. The seroprevalence largely depended on the age of the flocks. Seroprevalence was higher within the older pullet and layer flocks (83%-100%) than in the younger broiler flocks (0%-83%). In essence, all flocks older than 6 to 8 wk became infected. This is the first report of serologic evidence of CIAV in Subsaharan Africa. Since Southwest Nigeria is the main port of entry of imported chicken and the hub of major poultry breeders, the disease can probably be found throughout the country and beyond. Further studies are necessary to assess economic losses due to CIAV and the cost benefit of countermeasures.

An age-structured mathematical model of measles transmission in a vaccinated population is used to simulate the shift from a population whose immunity is derived from natural infection to a population whose immunity is vaccine-induced. The model incorporates waning of immunity in a population of vaccinees that eventually will become susceptible to a milder form of vaccine-modified measles with a lower transmission potential than unvaccinated classical measles. Using current estimates of duration of vaccine-derived protection, measles would not be expected to re-emerge quickly in countries with sustained high routine vaccine coverage. However, re-emergence is possible to occur several decades after introduction of high levels of vaccination. Time until re-emergence depends primarily on the contagiousness of vaccine-modified measles cases in comparison to classical measles. Interestingly, in a population with a high proportion of vaccinees, vaccine-modified measles and classical measles would occur essentially in the same age groups. Although waning of humoral immunity in vaccinees is widely observed, re-emergence of measles in highly vaccinated populations depends on parameters for which better estimates are needed.

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.

Transgenic carrot plants were developed expressing a designer polyepitope combining tandem repeats of a protective loop-forming B cell epitope (H386-400) of the measles virus hemagglutinin protein with a human promiscuous, measles-unrelated T cell epitope (tt830-844). Despite the sensitivity of the loop conformation to its molecular environment, proper folding was confirmed by conformation-dependent monoclonal antibodies. The antibodies also reacted with the boiled antigen in Western blot. Immunisation of mice peritoneally with carrot plant extracts induced high titers of antibodies that crossreacted strongly with the virus. Furthermore, the sera neutralised field isolates of different geographic origins and genotypes in a modified plaque reduction neutralisation assay performed on CD150-transfected Vero cells. These results demonstrate that transgenic carrot plants can serve as an efficient expression system to produce highly immunogenic, randomly assembled polyepitope antigens. The combined features of the selected epitopes and the potential of the plant expression system may pave the way towards new vaccines against measles.

Despite recent National Immunization Days in Burkina Faso, the rural province of Houët reported >400 measles cases in 2001 (82% not vaccinated). Phylogenetic analysis of 58 measles virus field isolates plus the first sequences from the Democratic Republic of the Congo and the Republic of Congo are reported. All viruses were genotype B3, which is common in the region. In Houët, there were two geographically confined genetic variants, suggesting two independent importation events. Strain diversity in Houët (1.5%) and the Congos was limited in comparison with Ibadan, Nigeria (4.6%), where measles is endemic. Strain variability, assessed by heteroduplex mobility assay, confirmed these findings. Despite large local pools of susceptible persons even after several rounds of vaccination, the limited strain diversity suggests that parts of rural Burkina Faso may be moving from an endemic to an epidemic transmission pattern of measles virus.

Vaccination with the current live attenuated measles vaccine is one of the most successful and cost-effective medical interventions. However, as a result of persisting maternal antibodies and immaturity of the infant immune system, this vaccine is poorly immunogenic in children <9 months old. Immunity against the live vaccine is less robust than natural immunity and protection less durable. There may also be some concern about (vaccine) virus spread during the final stage of an eventual measles eradication program. Opinions may differ with respect to the potential threat that some of these concerns may be to the World Health Organisation goal of measles elimination, but there is a consensus that the development of new measles vaccines cannot wait. Candidate vaccines are based on viral or bacterial vectors expressing recombinant viral proteins, naked DNA, immune stimulating complexes or synthetic peptides mimicking neutralising epitopes. While some of these candidate vaccines have proven their efficacy in monkey studies, aerosol formulated live attenuated measles vaccine are evaluated in clinical trials.

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine-map the highly conserved hemagglutinin noose epitope (H379-410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus-neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729-735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild-type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent-exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent-hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.

Although edible vaccines seem to be feasible, antigens of human pathogens have mostly been expressed in plants that are not attractive for human consumption (such as potatoes) unless they are cooked. Boiling may reduce the immunogenicity of many antigens. More recently, the technology to transform fruit and vegetable plants have become perfected. We transformed carrot plants with Agrobacterium tumefaciens to generate plants (which can be eaten raw) transgenic for an immunodominant antigen of the measles virus, a major pathogen in man. The hemagglutinin (H) glycoprotein is the principle target of neutralizing and protective antibodies against measles. Copy numbers of the H transgene were verified by Southern blot and specific transcription was confirmed by RT-PCR. The H protein was detected by western blot in the membrane fraction of transformed carrot plants. The recombinant protein seemed to have a 8% lower molecular weight than the viral protein. Although this suggests a different glycosylation pattern, proper folding of the transgenic protein was confirmed by conformational-dependent monoclonal antibodies. Immunization of mice with leaf or root extracts induced high titres of IgG1 and IgG2a antibodies that cross-reacted strongly with the measles virus and neutralized the virus in vitro. These results demonstrate that transgenic carrot plants can be used as an efficient expression system to produce highly immunogenic viral antigens. Our study may pave the way towards an edible vaccine against measles which could be complementary to the current live-attenuated vaccine.

The live-attenuated measles vaccine is poorly immunogenic in infants because of immune suppressive maternal antibodies and immaturity of the infant's immune system. Selected peptides corresponding to sequential, subdominant B cell epitopes of measles virus (MV) glycoproteins have been shown to induce neutralizing and protective antibodies even in the presence of whole virus antibodies. Similar to polysaccharide-conjugate vaccines, which are highly effective in infants a peptide-conjugate vaccine against measles is proposed. Such a vaccine induces carrier-specific T cells, avoiding measles-specific Th2 cells associated with the risk of atypical measles. This article discusses the rationale of such a strategy and its future potential.

Vaccine-induced immunity against measles is less robust than natural immunity. Waning of immunity in vaccines may eventually require a revaccination of adults. Measles antigens expressed in plants have been shown to be antigenic and immunogenic both after invasive and oral vaccination. Strategies for the vaccination of adults, the potential of an oral measles vaccine produced in edible plants and the design of suitable antigens are discussed.

Natural or wild-type (wt) measles virus (MV) infection in vivo which is restricted to humans and certain monkeys represents an enigma in terms of receptor usage. Although wt MV is known to use the protein SLAM (CD150) as a cell receptor, many human tissues, including respiratory epithelium in which the infection initiates, are SLAM negative. These tissues are CD46 positive, but wt MV strains, unlike vaccinal and laboratory MV strains, are not thought to use CD46 as a receptor. We have identified a novel CD46 binding site at residues S548 and F549, in the hemagglutinin (H) protein from a laboratory MV strain, which is also present in wt H proteins. Our results suggest that although wt MV interacts with SLAM with high affinity, it also possesses the capacity to interact with CD46 with low affinity.

Canine distemper virus (CDV) has a wide host spectrum, and during the past years, distemper has been observed in species that were previously not considered to be susceptible. In this study, we investigated the prevalence of CDV-specific antibodies in red foxes (Vulpes vulpes) sampled between May and November 1997. About 9 to 13% of the Luxembourg red fox population is positive for antibodies against CDV. Thus a sizeable proportion of red foxes has been exposed to CDV in the wild. The significance of CDV in red foxes is discussed.

Co-evolving mechanisms of immune clearance and of immune suppression are among the hallmarks of measles. B cells are major targets cells of measles virus (MV) infection. Virus interactions with B cells result both in immune suppression and a vigorous antibody response. Although antibodies fully protect against (re)infection, their importance during the disease and in the presence of a potent cellular response is less well understood. Specific serum IgM appears with onset of rash and confirms clinical diagnosis. After isotype switching, IgG1 develops and confers life-long protection. The most abundant antibodies are specific for the nucleoprotein, but neutralizing and protective antibodies are solely directed against the two surface glycoproteins, the hemagglutinin and the fusion protein. Major neutralizing epitopes have been mapped mainly on the hemagglutinin protein with monoclonal antibodies, producing an increasingly comprehensive map of functional domains.

Isolates of hepatitis B viruses were collected from 20 acute and chronic hepatitis patients in a highly endemic region of Nigeria. Sequencing classified the isolates to the ayw4, as they all contained the amino acid variations characteristic for that serotype. In the pre-S2 region of five isolates, three to seven amino acids were deleted, suggesting that immune escape mutations previously associated only with chronic HBV infection may be observed also in acute disease. Phylogenetic analysis of the complete pre-S2/S (large S) genes (831 nt) demonstrated that all the viruses belonged to the same genotype E. So far, no isolates of genotype E have been found in any other region of the world, including the Americas. This may suggest a relatively recent introduction of this genotype into humans and would explain the relatively low genetic diversity of viruses belonging to this genotype. One genotype E virus had been found previously in a chimpanzee, and viruses belonging to the CHIMP genotype are related to other genotype E viruses. These findings are compatible with a transmission of genotype E viruses from chimpanzees to humans., (Copyright 2001 Wiley-Liss, Inc.)

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

Life attenuated measles vaccines have dramatically reduced measles morbidity and mortality world-wide. Despite high vaccination coverage, measles outbreaks continue to occur both in developed and developing countries. While secondary vaccine failure may be responsible for disease in some seroconverted individuals, evidence suggests that many more vaccinees who are protected against disease may not be fully protected against virus infection. In low-income developing countries protection by maternal antibodies seems to erode faster than previously estimated especially in infants who were born to vaccinated mothers. Problems of infectivity and susceptibility of vaccinees will be compounded in case wild-type viruses become less sensitive to vaccine induced immunity. These observations suggest that elimination may be more easily achieved as long as large proportions of populations are protected by wild-type virus-induced immunity.

Peptides representing epitopes of the measles virus glycoproteins have been designed to induce neutralizing and protective antibodies. Those that escape recognition by passively acquired anti-whole virus antibodies could potentially be used as components of a 'pre-vaccine' that could be given during early childhood irrespective of persisting maternal antibodies. Unlike vaccines based on recombinant proteins, epitope-based vaccines can be designed to be compatible with a subsequent boost with the standard life attenuated vaccine. Although synthetic peptides may induce only short-term immunity they have the potential to close in young infants the gap of vulnerability until the standard live attenuated vaccine can be given at 9 or 15 months of age.

The different measles virus genotypes are confined to more or less distinct geographic regions. Molecular characterization of virus isolates has been successfully used to determine epidemiological links between cases and the geographic origin of imported viruses. In Europe, indigenous measles has been eliminated in some countries, but in others the disease is still endemic. Intra-outbreak variability can be used to differentiate between sporadic endemic cases and a 'pseudo-outbreak' of unrelated imported cases. The interruption of virus circulation by mass vaccination campaigns could be demonstrated by comparing the variability of pre-campaign viruses with post-campaign isolates. Simplified tools are being developed that could bring genotyping within reach of laboratories that do not have the possibility of sequencing.

The most exciting potential of phage displayed peptide libraries is to obtain small peptide molecules that mimic an antigen, at least with respect to a particular epitope. In addition to their interest as research tools, such mimotopes could in principle be useful as diagnostic tools or for eliciting antibodies to a predefined epitope. However, the reduction of the phage insert sequence to a short peptide that can compete with the antigenic and in particular with the immunogenic properties of the natural antigen faces considerable difficulties. This review assesses critically the antigenicity of phage displayed peptides as free peptides and in different molecular environments. The difficulties to use mimotopes to induce antibodies that bind to the natural antigen (crossreactive immunogenicity) and the considerable discrepancy between antigenicity and immunogenicity of phage-derived peptides are discussed. Peptides selected with antibodies from phage displayed random peptide libraries have raised considerable expectations as low molecular weight substitutes of the natural antigen. This review will focus on the results of phage displayed random peptide libraries screened with antibodies specific for proteins, carbohydrates and nucleic acids and critically examine how the above expectations have been met.

The neutralization capacity of sera from Luxembourgian adolescent vaccinees and from Nigerian women with measles-induced immunity to a number of measles virus strains was compared. Although both cohorts were matched for their hemagglutination inhibition and standard neutralization titers, 12 of the 22 late convalescent sera, and only 6 of 24 vaccinees neutralized all viruses. Similarly, only 2 of 20 viruses were not neutralized by at least 75% of late convalescent sera, in comparison to 10 of 20 viruses that resisted neutralization by at least 75% of the vaccinees. The more resistant viruses were not limited to a certain clade. One Nigerian virus was resistant to neutralization by 30% of the late convalescent women and by 75% of vaccinees. These results suggest that qualitative differences in neutralizing antibodies may reduce further protection of infants by passively acquired immunity against wild-type viruses when vaccinated girls become mothers., (Copyright 2000 Wiley-Liss, Inc.)

To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5-45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830-844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L(4)T(4))(2) and (L(2)T(2))(4)] and the helix conformation in one [(H(2)T(2))(4)] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L(2)T(2))(4), were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L(4)T(4))(2), (L(2)T(2))(4), (H(2)T(2))(4)] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.

Background: In developing countries vaccination against measles virus (MV) is generally administered at 9 months of age, although it is well-documented that protection of most infants by passively acquired maternal MV antibodies is waning before immunization is given. The purpose of this study was to investigate the decay of maternally derived MV antibodies in Nigerian infants as well as to compare a German and Nigerian cohort of paired mothers and newborns regarding the placental transfer efficiency of MV-specific IgG and total IgG antibodies., Methods: MV-specific IgG antibodies were measured with a commercially available MV-enzyme-linked immunosorbent assay, a recombinant hemagglutinin enzyme-linked immunosorbent assay as well as a neutralization assay. Total IgG values were determined with a standard immunoturbidimetric test., Results: Anti-MV IgG titers were twice as high in German newborns as in Nigerian newborns. An increased concentration of immunoglobulins transferred via the placenta was found only in the German cohort. High concentrations of total maternal IgG reduced the concentration of MV-specific as well as total IgG that crossed the placenta. Furthermore only 17% of the 4-month-old Nigerian infants were still protected against measles. Antibodies had a biologic half-life of 33 days and a biochemical half-life of 48 days., Conclusions: Our findings demonstrate that the decay of passively acquired MV antibodies occurred even more rapidly than expected resulting in susceptibility to MV in most of the 4-month-old infants in Nigeria. Furthermore transfer of maternal anti-MV IgG and total IgG antibodies to the newborn was more efficient in the German cohort compared with the Nigerian group. These findings suggest the use of alternative vaccination strategies in developing countries to possibly reduce the window of susceptibility against measles.

From March to July 1996 a measles outbreak occurred in northern Luxembourg with 110 reported cases centered around two primary schools (85 cases) and the surrounding community (25 cases). Eighty four suspected cases were confirmed serologically. Vaccine coverage was estimated from questionnaire-based surveys at the two primary schools to be 70 and 76%, respectively. Vaccine efficacy during the outbreak was estimated to be 94.6% [95% confidence interval (CI) 90.4-97.0]. Using the information from the, school surveys, we obtained estimates of the basic reproduction number of measles of 7.7 (95% CI 4.4-11.0) and 6.2 (95% CI 3.5-8.9), respectively. Assuming a 95% vaccine efficacy, these estimates correspond to minimal vaccine coverages of 91.6% (95% CI 81.4-95.7) and 88.3% (95% CI 75.5-93.4) which would have been necessary to minimize the chances of a major outbreak occurring. We can confirm that major outbreaks in similar school settings can only be prevented if vaccination coverage exceeds 90%.

The sequence H379-410 of the measles virus haemagglutinin (MV-H) protein forms a surface-exposed loop and contains three cysteine residues (Cys-381, Cys-386 and Cys-394) which are conserved among all measles isolates. It comprises the minimal sequential B cell epitope (BCE) (H386-400) of the neutralizing and protective MAb BH6 that neutralizes all wild-type viruses tested. The aim of this study was to design synthetic peptides which induce neutralizing antibodies against MV wild-type isolates. Peptides containing one or two copies of T cell epitopes (TCE) and BCEs of different lengths (H386-400, B(CC); H379-400, B(CCC)), in different combinations and orientations were produced and iteratively optimized for inducing neutralizing antibodies. Peptides with the shorter BCE induced sera that cross-reacted with MV but did not neutralize. The longer BCE containing the three cysteines (B(CCC)) and two homologous TCE were required for neutralization activity. These sera neutralized wild-type strains of different clades and geographic origins. Neutralizing serum was also obtained after immunization with human promiscuous TCEs. Furthermore B(CCC)-based peptides were fully immunogenic even in the presence of pre-existing MV-specific antibodies. The results suggest that subunit vaccines based on such peptides could potentially be used to actively protect infants against wild-type viruses irrespective of persisting maternal antibodies.

During the WHO campaign to eradicate measles, accurate discrimination between immune and non-immune individuals will become increasingly important. Due to waning immunity in vaccinated populations, the performance of a measles IgG assay depends mainly on its ability to detect reliably seronegative individuals among many vaccinees with low antibody levels. New serological tests based on recombinant proteins detect only a fraction of the total measles virus (MV) specific antibodies. Therefore, several assays based on recombinant MV-haemagglutinin (ELISA and flow cytometry) or MV-fusion protein (flow cytometry) as well as neutralisatlon and haemagglutination test have been evaluated using a large panel of low-titre and negative sera. Since such an evaluation is highly dependent on threshold values for positivity, the receiver operating characteristic curve analysis was applied. The H-FACS and the H-ELISA showed the best performing characteristics (specificity: 97.4 and 96.1%, respectively; sensitivity: 88.1 and 89.6%, respectively) and may be an alternative to the neutralisation assay. The number of undefined/grey zone sera was significantly lower compared to a commercial whole virus-based ELISA and therefore fewer individuals would be vaccinated unnecessarily.

Phage-displayed peptide libraries represent an efficient tool to isolate peptides that bind a given target molecule. After several selection rounds, generally a large pool of target binding phages is obtained. Conventional analysis of the selected phage population involves extensive sequencing of many clones, most of which can be identical. We have adapted the Heteroduplex Mobility Assay (HMA) for pre-screening of phage inserts that were amplified by direct colony PCR of ELISA-positive clones. This strategy allowed for the rapid and reproducible assignment of insert sequences to different 'heteroduplex migration groups'. Sequence analysis of only one representative of each HMA migration group then completes the characterisation of the binding phage population. In our model experiments, only 16% of HMA pre-screened clones required further sequence analysis.

In Europe measles incidence remains high and in some parts the disease is likely to be still endemic due to insufficient vaccination. Luxembourg experienced an outbreak with at least 110 cases in 1996, and cases continued to be reported throughout 1997. We used molecular epidemiology to investigate this apparent endemicity. On the basis of their N gene sequences, the isolates were assigned to the typical European C2 and D6 genotypes. Sequence diversity within the outbreak was 0.2%. The nucleotide distance between the C2-viruses of the outbreak and the other C2 isolates was at least three or four times higher, suggesting an independent origin of the latter viruses. Similarly, the four D6 viruses found in Luxembourg were thought to be of at least two or three origins. Thus, we propose here to use intra-outbreak sequence diversity to differentiate between sporadic endemic cases and a "pseudo-outbreak" of multiple unrelated imported cases.

The isolation of 98/ASF/NG, a strain of African Swine Fever Virus (ASFV) associated with a 1998 epizootic in Nigeria, is reported. This first isolate of the virus from West Africa was identified through a successful polymerase chain reaction (PCR) amplification and sequencing of a 280 base pair (bp) fragment of the Major Capsid Protein (VP72) gene. Further amplification and sequence analysis of a 1.9 kilobase pair (kbp) fragment encompassing the complete VP72 gene showed that the isolate has a 92.2%, 92.4%, and 97.2% homology with previously sequenced Ugandan, Dominican Republican and Spanish isolates respectively. Of the 50 nucleotide changes observed in this highly conserved gene, 45 were found to result in 40 amino acid changes clustered around the central region (position 426 to 516) of the VP 72 protein while changes at the remaining 5 positions were silent. These changes also led to the loss of two out of the seven potential N-glycosylation sites which are in this gene conserved among all isolates. The possible epizootiological implications of such mutations in a highly conserved gene of a DNA virus is discussed in relation to this outbreak.

An increasing body of evidence suggests that a substantial proportion of individuals who respond to measles vaccine display an antibody boost accompanied by mild or no symptoms on exposure to wild virus. It is unknown whether this emerging class of individuals can support transmission. The epidemiologic consequences of vaccinated individuals able to transmit virus are investigated using a mathematical model. Parameters for this model are estimated using regression analysis on a Canadian serologic data set. The authors confirm that neutralizing antibodies are decaying significantly in absence of circulating virus. Based on a protective threshold plaque reduction neutralization (PRN) titer of 120, the authors estimate the mean duration of vaccine-induced protection in absence of reexposure to be 25 years (95% confidence interval (CI) 18, 48). After long-term absence of circulating virus, the mathematical model predicts that 80% (95% CI 65, 91) of all seroconverted vaccinees have titers below the protective threshold. In this case, elimination of measles virus cannot be achieved by a single-dose routine vaccination strategy if the basic reproduction number in vaccinated individuals exceeds 1.24 (95% CI 1.10, 1.53). For this reason, there is a need to establish the intensity and duration of infectiousness in vaccinated individuals.

The sequence H236-256 of the measles virus (MV) hemagglutinin (H) contains the sequential epitope of the neutralizing and protective monoclonal antibody (mAb) BH129 with the minimal epitope E(245)L-QL(249). Using this mAb, we have recently developed 7mer mimotopes binding up to 135x better than the corresponding 7mer epitope H244-250. In this study, we combined T cell epitopes (TCE) with either highly crossreactive 7mer mimotopes, 13mer mimotopes or less crossreactive MV-derived B cell epitopes (BCE). Antigenicity of these TBB, TTB and TTBB peptides was determined with BH129 in a competition ELISA against MV. We found that chimeric peptides including mimotopes were up to 80x better binders to the mAb than peptides containing the original BCEs. All peptides irrespective of their antigenicity were used for immunization to compare their virus- crossreactive immunogenicity. Unexpectedly, none of the highly antigenic mimotope-based peptides induced MV-crossreactive antibodies. In contrast, a number of peptides with the viral BCE sequence that did not bind to the mAb, induced MV-crossreactive and even neutralizing antibodies. This report describes a striking example of disparity between antigenicity and crossreactive immunogenicity and casts considerable doubt on the predictive value of antigenicity in immunogenicity studies, considerably complicating the selection of potential vaccine candidates.

A comprehensive phylogenetic study based on the hemagglutinin (H) protein of all known African measles virus (MV) isolates is presented. The study includes 64 new H gene sequences from Ghana. Nigeria and South Africa as well as viruses from Zambia and The Gambia for which only incomplete sequencing data were available and that have previously not been genotyped. The results provide further support to the tentative assignment of the Nigerian and Ghanaian viruses to a new genotype B3 within clade B. A distinct geographic distribution pattern emerged with clade B viruses circulating exclusively in African countries north of the equator. All MV strains from southern Africa grouped in clades A and D with the majority of viruses belonging to genotype D4. The viruses considerably differed by their sensitivity to neutralization by monoclonal antibodies (mAb), but three selected antibodies were sufficient to distinguish between African MVs representing four different genotypes.

The neutralizing and protective monoclonal antibody BH47 defines the sequential epitope H236-255 of the measles virus hemagglutinin protein (MV-H). The objective of this study was to design peptides combining this B cell epitope (BCE) with different T cell epitopes (TCE) to obtain protective immunity. Most TTB peptides based on the 15mer BCE H236-250 induced MV-crossreactive antibodies, but only certain TCE induced virus neutralizing antibodies. The shortest BCE required for MV-reactivity and -neutralization was the 8mer H243-250 containing residue R243 implicated in CD46 down-regulation. Sera obtained after immunization with the TTB peptide containing the MV-derived TCE F421-435 protected mice against a lethal challenge with a neuro-adapted MV strain. Our results further demonstrate that this TTB peptide is fully immunogenic, even in the presence of protective levels of pre-existing MV-specific antibodies, suggesting that subunit vaccines based on such peptides could potentially be used to immunize infants in the presence of persisting maternal antibodies. It is therefore interesting that neutralizing antibodies were also obtained with a TTB peptide comprising a human promiscuous TCE (tt830). However, our results also emphasize the need to test sera induced with epitope-based vaccines against different virus strains, in particular if the epitope is not fully conserved.

Sub-Saharan Africa is one of the regions of the globe with the highest measles-related morbidity and mortality. Yet only seven virus isolates from this vast region have been phylogenetically characterized on the basis of their nucleoprotein, the last one in 1991. To characterize the prevalent wild-type viruses and to understand their circulation pattern, a large panel (n = 45) of isolates was collected in Ghana and Nigeria in 1997 and 1998. On the basis of their nucleoprotein sequence, the viruses clearly belong to clade B but a reshuffling of the structure of this clade was proposed, tentatively extending the number of genotypes from two to three on the basis of quantitative criteria. The sequences revealed the co-circulation of at least two distinct viruses in the cities of Lagos and Ibadan, suggesting that the number of susceptible individuals seems to be high enough to support endemic circulation of at least two distinct viruses. The endemic co-circulation of several viruses may well be a characteristic of communities with low vaccination rates. One of these viruses was also found in Accra in 1998 as well as in a 1994 case linked to distant Kenya, suggesting that clade B viruses are prevalent in sub-Saharan Africa while non-B viruses seem to dominate the south of Africa.

The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V- was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C- progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C--infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C- was as potent as MV tag or MV V- in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.

MHC class II molecules present to CD4+ T cells protein fragments which mostly derive from the extracellular and from the endosomal compartments. Determinants of cytosolic proteins are, however, also displayed by MHC class II molecules following pathways which are still not yet fully characterized. Here we describe the isolation of DRB1*1103-restricted T cell clones specific for the measles virus (MV) nucleoprotein peptide 185-199 (N185). Experiments were then conducted to delineate how this determinant is assembled with DR molecules. In vitro binding analyses indicated that complexes between the N185 peptide and DRB1*1103 protein are optimally constituted at pH 4-4.5. In cellular experiments it was observed that chloroquine, leupeptin and emetine, which are classical inhibitors of presentation of MHC class II-restricted antigens, when added during infection of B cells with MV, prevent presentation of the N185 determinant. In addition, it was found that the N185 determinant is efficiently presented when the nucleoprotein is exogenously provided to B cells, either by blocking MV fusion with the peptide FFG or by the use of purified nucleoprotein. In contrast, it was observed that nucleoprotein recombinant vaccinia virus (vv-N)-infected B cells weakly stimulated N185-specific T cells, indicating that the restricted localization of the nucleoprotein in the cytosol resulted in a poor presentation of the N185 determinant. Taken together, these findings suggest that it is prior to delivery of the nucleoprotein into the cytosol that the N185 determinant is efficiently assembled with newly synthesized DR molecules in the acidic environment of the endosomal compartment.

The loop comprising aminoacids H236-256, connects two strands of sheet 1 of the propeller-like hemagglutinin (H) protein of the measles virus (MV) and contains a putative active site residue (R253), a residue implicated in CD46-downregulation (R243) and the minimal epitope E245L-QL249 of the neutralising and protective monoclonal antibody BH129. The objective of this study was to design synthetic peptides which induce neutralising antibodies against this important functional domain. Peptide-design was based on the colinear synthesis of this sequential B cell epitope (BCE) with different T cell epitopes (TCE). Chimeric constructs were systematically optimised with respect to length and copy number of the BCE and the nature and orientation of the TCE. Surprisingly, the induction of MV-crossreactive antibodies did not correlate with the antigenicity of the peptides. The best MV-crossreactive antibodies were obtained with TB oriented constructs containing TCEs of the MV fusion (F) protein and the BCE H236-250 (TB15mer) or H236-255 (TB20mer). In vitro virus-neutralising sera were obtained solely with the latter construct. A glycine scan showed that binding to MV depended on a defined pattern of contact residues compatible with the putative alpha helical nature of this epitope. The contact residues of the neutralising serum (S244EL-QL249) differed from those of the non-neutralising serum (S244EL246) but no unique differences in the immunoglobulin subclasses were detected. Surface plasmon resonance measurements detected a higher affinity for the neutralising serum compared to the TB15mer serum. These results emphasize the need of an optimal design of immunogenic peptides which cannot always be guided by the antigenicity of the peptide constructs. This study demonstrates that neutralising antibodies can be generated with peptides mimicking this helical epitope, provided that the critical contact residues are recognized with high affinity and underlines the potential of the epitope as an element of a future subunit vaccine.

Simplified tests based on recombinant antigens are considered to be important for monitoring immunity against measles virus (MV). The hemagglutinin protein (H) is the main target for neutralising and protective antibodies. We produced a recombinant MV-H protein, in a high-yield mammalian expression system based on the Semliki Forest virus replicon. The antigenicity of this recombinant protein was investigated with monoclonal antibodies and its suitability for measuring the immune status of vaccinees was tested in a large cohort by ELISA (H-ELISA). The results were evaluated against neutralisation (NT) and hemagglutination inhibition (HI) titers and MV-specific IgG measured in a commercial whole-virus based ELISA (MV-ELISA, Enzygnost). The H-ELISA correlated better with HI (r=0.78) and NT titers (r=0.80), than the MV-ELISA (HI, r=0.58; NT, r=0.59). In contrast to the MV-ELISA, the H-ELISA detected no false-positive sera (P < 0.02) and the number of false-negative sera was significantly lower in the H-ELISA than in the MV-ELISA (4/378 vs. 15/378; P < 0.025). The performance of the H-ELISA did not deteriorate significantly when, instead of background corrected net values, uncorrected raw O.D. values of the H-antigen were considered, or when early time points (30 min) were evaluated. These results demonstrate that the recombinant H-ELISA detects efficiently non-immune individuals among vaccinees, despite their relatively low MV-antibody levels. A simplified format with single value measurements did not result in loss of sensitivity or specificity and its performance compared favorably with commercial ELISAs based on whole virus.

Serological evidence indicates that measles virus (MV) could circulate in seropositive, fully protected populations. Among individuals fully protected against disease, those prone to asymptomatic secondary immune response are the most likely to support subclinical MV transmission. The serological characteristics of protected subjects who developed secondary immune response after reexposure to measles have been described recently [Huiss et al. (1997): Clinical and Experimental Immunology 109:416-420]. On the basis of these data, a threshold of susceptibility was defined to estimate frequencies of secondary immune response competence in different populations. Among measles, late convalescent adults (n = 277) and vaccinated high school children (n = 368), 3.2-3.9% and 22.2-33.2%, respectively, were considered susceptible to secondary immune response. A second vaccination did not seem to lower this incidence. Even when estimates of symptomatic secondary immune response (e.g., secondary vaccine failure) were taken into account, susceptibility to subclinical secondary immune response was still 5-8 times higher after vaccination than after natural infection. Although viral transmission between protected individuals has never been directly demonstrated, the data describe a population in which protected but infectious persons could potentially be of epidemiological importance.

Antigenicity and conformational propensities of synthetic peptides corresponding to the sequential epitope H236-255 of the measles virus hemagglutinin protein were investigated. This epitope corresponds to the neutralising and protective monoclonal antibody BH129 and includes Arg243, implicated in CD46-down-regulation and Arg253 that has been mapped to the putative enzymatic site. Fine mapping with truncation-, elongation-, Gly- and Ala-substitution analogues defined EL-QL as the critical residues of the minimal epitope S244ELSQL249. CD spectra of peptides, comparison with the 3D-structure of homologous sequences, and prediction algorithms suggested a helical structure with the contact residues E245L-QL249 located on the protein surface. Mimotopes obtained with a 6-mer phage display library contained a consensus Pro (important for binding) instead of Ser247 of the wild-type sequence (irrelevant for binding). The kink induced by Pro seemed to be essential to bring the 4 contact-residues in the mimotopes and in the corresponding short peptides together. CD analysis and prediction algorithms suggested that non-helical conformations of the phage insert and of the peptides may favourably mimic the antigenic helical turns of the wild-type sequence, resulting in an up to 135 times higher antigenicity of the mAb towards the mimotope peptides.

Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (R2 = 0.66), hemagglutination inhibition test (HI) titers (R2 = 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA; R2 = 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (R2 = 0.52) or HI (R2 = 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as "gold standards." In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively; P was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (n = 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively; P < 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for surveillance for immunity to measles and that such an assay could be more efficient in detecting susceptibility to measles. Furthermore, unlike whole MV-based antigens, H-protein would also be suitable for use in the development of a simple field test for the diagnosis of measles.