Biomedical subjects

C P Muller

Publications and source records attributed to C P Muller.

At least 37 records · Page 2Linked to original sources

PubMed2000

Evaluation of different measles IgG assays based on recombinant proteins using a panel of low-titre sera.

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.

Antibodies, Viral↗

PubMed2000

Estimation of the basic reproduction number of measles during an outbreak in a partially vaccinated population.

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%.

Adult↗

PubMed2000

An outbreak of African Swine Fever in Nigeria: virus isolation and molecular characterization of the VP72 gene of a first isolate from West Africa.

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.

African Swine Fever↗

PubMed2000

Heteroduplex mobility assay (HMA) pre-screening: an improved strategy for the rapid identification of inserts selected from phage-displayed peptide libraries.

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.

Animals↗

PubMed2000

Placental transfer and decay of maternally acquired antimeasles antibodies in Nigerian children.

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.

Adult↗

PubMed2000

Neutralization of measles virus wild-type isolates after immunization with a synthetic peptide vaccine which is not recognized by neutralizing passive antibodies.

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.

Amino Acid Sequence↗

PubMed2000

Expression of measles virus antigens in Streptococcus gordonii.

The measles virus proteins haemagglutinin (HA) and fusion protein (F), which together mediate attachment and penetration of the virus in the host cell and can elicit production of neutralising antibodies in the course of natural infection were expressed in the vaccine vector Streptococcus gordonii, a Gram-positive bacterium normally present in the human oral cavity. HA and F were expressed as fusion proteins attached to the bacterial surface, and were both found to be immunogenic when the recombinant S. gordonii were inoculated subcutaneously in mice.

Animals↗

PubMed1999

Modeling the impact of subclinical measles transmission in vaccinated populations with waning immunity.

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.

Adolescent↗

PubMed1999

A hemagglutinin-derived peptide-vaccine ignored by virus-neutralizing passive antibodies, protects against murine measles encephalitis.

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.

Animals↗

PubMed1999

Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates.

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.

Africa↗

PubMed1999

Crossreactivity of mimotopes and peptide homologues of a sequential epitope with a monoclonal antibody does not predict crossreactive immunogenicity.

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.

Animals↗

PubMed1999

Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa.

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.

Base Sequence↗

PubMed1999

Nonstructural C protein is required for efficient measles virus replication in human peripheral blood cells.

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.

Animals↗

PubMed1998

Estimated susceptibility to asymptomatic secondary immune response against measles in late convalescent and vaccinated persons.

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.

Adolescent↗

PubMed1998

Enhanced antigenicity of a four-contact-residue epitope of the measles virus hemagglutinin protein by phage display libraries: evidence of a helical structure in the putative active site.

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.

Animals↗

PubMed1998

The molecular basis of virus crossreactivity and neutralisation after immunisation with optimised chimeric peptides mimicking a putative helical epitope of the measles virus hemagglutinin protein.

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.

Amino Acid Sequence↗

PubMed1998

A simplified immunoassay based on measles virus recombinant hemagglutinin protein for testing the immune status of vaccinees.

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.

Adolescent↗

PubMed1998

Processing of the DRB1*1103-restricted measles virus nucleoprotein determinant 185-199 in the endosomal compartment.

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.

Amino Acid Sequence↗