Persistence of measles neutralizing antibody related to vaccine and natural infection acquired before HIV infection.

Little is known about long-lasting measles protective immunity when exposure to wild-type or vaccine measles virus precedes HIV infection. The results obtained suggest that measles immunity wanes and the lowest measles geometric mean titres (GMT) were significantly associated with measles vaccine-induced immunity in individuals that later developed HIV infection (86% prevalence, GMT 164 mIU/ml) compared to naturally induced immunity in HIV-infected adults (100% prevalence, GMT 340 mIU/ml, P = 0·0082) or non-HIV infected adults (100%, GMT 724 mIU/ml, P = 0·0001), and vaccine-induced immunity in non-HIV-infected adults (100%, GMT 347 mIU/ml, P = 0·017). The study was conducted in an area without wild-type virus circulation since 2000. The absence of virus circulating may alter the paradigm of lifelong immunity to measles virus after vaccination. As the proportion of HIV-infected individuals possessing only vaccine-induced immunity continues to grow, checking the status of measles immunity in this group is strongly recommended.

[Distribution of genotypes of hepatitis C in the city of Córdoba, Argentina]. / Distribución de genotipos de hepatitis C en la ciudad de Córdoba, Argentina.

BACKGROUND: Knowledge of Hepatitis C virus genotype (HCV) present in a patient has an epidemiological interest. In addition, it has an important prognostic value that guides the duration and success of treatment. AIMS: To analyze the distribution of genotypes in HCV-positive patients and linking them with the viral load before and after treatment, evaluating sustained viral response. PATIENTS AND METHODS: We retrospectively analyzed the results of genotyping and HCV viral load of 71 patients during the period January 2001 to May 2009. The genotypes were determined by RFLP (restriction fragment length polymorphism) and the viral load by NASBA HVC quantitative. Statistical analysis was performed using the Infostat program. RESULTS: 59% of patients were women. The frequency of genotypes was: 39% type 1, 58% type 2 and 3% type 3. We do not find a cutoff value of viral load to identify the different genotypes, although patients with genotype 1 had a higher number of viral copies than those of genotype 2 (p <0.0001). After treatment, 95% of patients with genotype 2 had a sustained viral response versus 67% of patients with genotype 1. CONCLUSIONS: The genotype 2 was the most prevalent in our population. It also confirmed the impact of knowledge of HCV genotype on sustained viral response, which was related related surgical interventions to infection with HCV type 2.

Evaluation of antibodies against a rubella virus neutralizing domain for determination of immune status.

The protective immune responses against rubella virus (RV) are related to its neutralizing epitopes, an issue that is important to consider when assessing the immune status of patients with remote infection. In the present paper, we compare the antibodies detected by a synthetic-peptide-based enzyme immunoassay (EIA) with antibodies detected by the traditional technique of hemagglutination inhibition (HIA) in patients with remote RV infection. The synthetic peptide used as an antigen (SP15) represents a neutralizing epitope that corresponds to amino acids 208 to 239 of the E1 glycoprotein. The SP15-EIA was developed, all variables that affected the assay were standardized, and the test was validated using reference sera. Serum samples (n = 129) from patients with remote RV infection were tested by HIA and SP15-EIA. Discrepant sera were assayed by MEIA (IMX/Abbot). The comparison between HIA and SP15-EIA, taking HIA as the standard methodology for determining immune status, showed that SP15-EIA is very specific and sensitive for detecting protecting antibodies (specificity, 100%; sensitivity, 98.20%). This study demonstrates that antibodies against the neutralizing domain represented by SP15 would be important in the memory response after natural infection and may be a good tool in the determination of the true immune status of patients with remote infection with regard to RV.

Neutralizing monoclonal antibody to the E1 glycoprotein epitope of rubella virus mediates virus arrest in VERO cells.

The best-known mechanism of action of antibody-mediated virus neutralization is to impede the entrance of viruses to host cells, as determined by neutralization assays. Antibodies may also inhibit the exit of rubella virus (RV) from infected host cells; in this case, the interaction of the antibodies with their domains must occur on the plasma membrane, because antibodies cannot enter the cells. In the present study, we were able to block temporally the exit of virions from RV-infected cells by the binding of monoclonal antibody (mAb) H3 to their surface. The objective was accomplished in three steps: first, we determined the duration of the viral replication cycle; then we established the kinetics of the presence of the domains defined by our mAbs in the cytoplasm of RV-infected VERO cells; and, finally, we assessed the release of viral particles to the supernatant of infected VERO cells in the presence or absence of mAbs or positive and negative mice sera. RV-specific mice sera and mAb H3, which binds to the amino acid sequence 208-239 of the RV-E1 glycoprotein, were able to delay for 24 hours the release of virions from infected cultures, suggesting that the reaction of mAb H3 with its epitope may arrest any change necessary for the assembly and/or release of virions. In conclusion, the neutralizing domain recognized by mAb induces antibodies that can block the viral replication by several mechanisms of action, such as the obstruction of virus entry into cells and the delay of viral release. All of these mechanisms are intimately involved in the critical virus-host cell interactions that allow self-limitation of the infection.

Analysis of viral glycoproteins by glycosidic digestion inside a polyacrylamide gel.

We adapted the method described by Cleveland et al. (1977); (Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106) to study the glycosidic residues linked to the viral glycoproteins of two enveloped viruses: Junin virus (JV) and rubella virus (RV). Radioiodinated glycoproteins were obtained from purified virions, isolated from SDS-polyacrylamide gels and then hydrolysed by specific glycosidases inside a second gel. N-linked oligosaccharides, mannose and galactose were found as terminal residues in the JV-G1 glycoprotein. Mannose and N-glycans of complex hybrid type were present on RV glycoproteins.

Cell-fusion assay for the detection of rubella virus in Vero cells.

BACKGROUND AND OBJECTIVES: Rubella virus (RV) produces a subtle and slow-developing cytopathic effect in Vero cells that is difficult to recognize, especially at low multiplicities of infection. In order to facilitate the detection of RV in cell culture, we standardized a low-pH virus-mediated cell-fusion assay. STUDY DESIGN: The incubation periods, temperatures, pH and multiplicity of infection were established. The specificity of the method was tested by immunofluorescence assay and cell-fusion inhibition by specific sera. RESULTS: Six days post infection, Vero cells were treated for 5 min with fusion medium. After that, monolayers were incubated with medium at neutral pH for 16 h and then stained. Gigantic cells with multiple nuclei were observed. CONCLUSIONS: The method allowed the observation of unequivocal images that are easier to recognize than the cytopathic effect caused by RV in the same cell line. At the same time, the method is simple, accessible and shown to be specific to demonstrate the replication of several strains and isolates of RV in Vero cells.

Presence of a neutralizing domain in isolates of rubella virus in Cordoba, Argentina.

We studied the presence of a neutralizing epitope of rubella virus (RV) in locally circulating strains in Cordoba, Argentina, using binding by the monoclonal antibody (MAb) H3. This epitope is contained in a sequence of the E1 glycoprotein (E1208-239) represented by the synthetic peptide SP15. H3 MAb showed specific binding to SP15 by enzyme-linked immunosorbent assay (ELISA). One wild-type postnatal isolate, four clones derived from this isolate, and one congenital isolate were reactive with H3 by ELISA. These results suggest that the region of RV represented by SP15 is a domain present in locally circulating strains.

Detección de anticuerpos para rubéola en muestras de sangre recolectadas por filtro / Detection of rubella virus antibodies in whole blood collected on filter paper

El síndrome de rubéola congénita (CRS) puede ser fácilmente evitado si las mujeres en edad gestacional conocen su estado inmune y, en caso de ser seronegativas, reciben la vacunación previa al embarazo. Con el objetivo de determinar el estado inmune de grandes poblaciones se adoptó un método sencillo para la recolección y el almacenamiento de las muestras de sangre. A un total de 100 pacientes se les extrajo suero y sangre que fue absorbida en un círculo de papel de filtro, que capta aproximadamente 0,1 ml de suero. Fueron analizados los títulos de anticuerpos inhibidores de la hemaglutinación (TIH). En las muestras de papel, luego de ser almacenada en bolsas plásticas a 25 grados centígrados durante 7, 14, 21, 30, 60 y 100 días, con respecto a los sueros de cada paciente. De las muestras de sangre obtenidas en el papel de filtro a los 30 días de realizada la extracción, el 72 o/o tuvo el mismo TIH y el resto poseía una dilución de diferencia en relación con los sueros correspondientes. De las muestras ensayadas a los 60 días de extracción un 59 o/o tuvo el mismo TIH y el 41 o/o una dilución de diferencia. A los 100 días de extracción, el 51 o/o de las muestras tuvieron el mismo TIH, el 38 o/o una dilución de diferencia y el 11 o/o tuvieon 1 o más de una dilución de diferencia. En conclusión, los resultados de ese estudio mostraron que las muestras de sangre recolectadas sobre papel de filtro pueden ser usadas para la detección de anticuerpos antivirus rubéola determinados por IHA dentro de los 30 días post-obtención con una sensibilidad y especificidad del 100 o/o. Esta muestra en papel es estable a temperatura ambiente, sin ninguna restricción de aire o esterilización especial (AU)

Detección de anticuerpos para rubéola en muestras de sangre recolectadas por filtro / Detection of rubella virus antibodies in whole blood collected on filter paper

El síndrome de rubéola congénita (CRS) puede ser fácilmente evitado si las mujeres en edad gestacional conocen su estado inmune y, en caso de ser seronegativas, reciben la vacunación previa al embarazo. Con el objetivo de determinar el estado inmune de grandes poblaciones se adoptó un método sencillo para la recolección y el almacenamiento de las muestras de sangre. A un total de 100 pacientes se les extrajo suero y sangre que fue absorbida en un círculo de papel de filtro, que capta aproximadamente 0,1 ml de suero. Fueron analizados los títulos de anticuerpos inhibidores de la hemaglutinación (TIH). En las muestras de papel, luego de ser almacenada en bolsas plásticas a 25 grados centígrados durante 7, 14, 21, 30, 60 y 100 días, con respecto a los sueros de cada paciente. De las muestras de sangre obtenidas en el papel de filtro a los 30 días de realizada la extracción, el 72 o/o tuvo el mismo TIH y el resto poseía una dilución de diferencia en relación con los sueros correspondientes. De las muestras ensayadas a los 60 días de extracción un 59 o/o tuvo el mismo TIH y el 41 o/o una dilución de diferencia. A los 100 días de extracción, el 51 o/o de las muestras tuvieron el mismo TIH, el 38 o/o una dilución de diferencia y el 11 o/o tuvieon 1 o más de una dilución de diferencia. En conclusión, los resultados de ese estudio mostraron que las muestras de sangre recolectadas sobre papel de filtro pueden ser usadas para la detección de anticuerpos antivirus rubéola determinados por IHA dentro de los 30 días post-obtención con una sensibilidad y especificidad del 100 o/o. Esta muestra en papel es estable a temperatura ambiente, sin ninguna restricción de aire o esterilización especial

[Detection of rubella antibodies in blood samples collected on filter paper]. / Detección de anticuerpos para rubéola en muestras de sangre recolectadas en papel de filtro.

Congenital rubella syndrome (CRS) could be prevented if young women knew their immune status before pregnancy, contributing in this way to decrease the birth morbidity rate due to CRS among the children. Our objective was to optimize the detection of rubella virus-antibodies by HAI, using an easier and safer method to collect samples of big populations. One hundred specimens, obtained from patients in a pediatric hospital and pregnant women in an institute of Virology were used for this work. Venous blood was drawn and collected in a test tube, and few drops were spotted onto filter paper circles. These samples were kept in envelopes and stored at room temperature until analysis. Seventy two percent of dried blood samples had titers identical to those of the corresponding serum samples, and 28% dried blood samples showed 1 dilution of difference. Storage of dried blood at room temperature for 30 days did not affect the HAI titers. Up to 60 days post attainment, 59% dried blood samples had identical titers to those of the corresponding serum samples, and 41% dried blood samples showed one dilution of difference. At 100 days of storage 51% dried blood samples had identical titers to those of the corresponding serum samples, 38% dried blood samples showed 1 dilution of difference and 11% and more than 1 dilution of difference. In conclusion, dried blood on filter paper is an easier method to transport and store blood samples for the determination of rubella virus immunity, for as long as 30 days. It could be used for large-scale epidemiological studies. The sensitivity and specificity of HAI performed on dried blood samples was 100%. Only 0.25 ml of whole blood is needed and the samples are stable at room temperature, without air or sterile conditioning. The proposed methodology is a practical approach to collect, transport and store blood samples. Moreover the blood dried on paper spots can be placed in a plastic bag and mailed to a reference laboratory. This is an appropriate alternative method for serological screenings in developing countries.

Different affinity of monoclonal antibodies for conserved neutralizing epitopes on two strains of rubella virus.

There is, apparently, only one serological type of rubella virus (RV) in the population, although several isolates exist with different characteristics. Some authors failed to detect significant differences among RV strains by neutralization, hemagglutination inhibition, and enzyme immunoassay using polyclonal and monoclonal antibodies, but differences in growth, plaque morphology, and temperature sensitivity between vaccine and wild-type strains were shown by Chantler et al. (3) With the purpose of analyzing the possible differences among several strains of RV, we studied the affinity constant of two monoclonal antibodies (MAbs) for two conserved neutralizing epitopes. Wild-type Cordoba (regional isolation of a post-natal infection) and RA 27/3 (vaccine) strains of RV were tested. H3 and H14 MAbs were generated against wild-type Cordoba strain. They defined two epitopes with conserved neutralizing and hemagglutinating activity on both strains. The affinity of the MAbs (expressed as the affinity constant), was greater for Cordoba strain than for RA 27/3. Analyzing the results obtained, we conclude that the neutralizing epitopes defined by our MAbs on E1 glycoprotein are conserved in the two strains, but react with significative different affinities. This could be a way to characterize antigenically different viral strains of the same serotype.

[Isolation of Chlamydia trachomatis and immune response in different populations]. / Aislamiento de Chlamydia trachomatis y respuesta inmune en diferentes poblaciones.

We studied the presence of C. trachomatis-specific IgG and IgM in adults and newborns, respectively, and attempted isolation of the bacteria in cell culture. The determination of antibodies was carried out by an IFA on C. trachomatis infected (L2 434/Bu serotype) McCoy cells, cultured in 24-well plastic plates. We found C. trachomatis-specific IgG in 27% of women with clinical symptoms, in 40% of women being attended for periodic gynecological control, in 60% of infertile women and in 10% of pregnant women. A proportion comparison test revealed the presence of specific IgG as highly significative for the group of infertile women as compared to the group of pregnant women (p < 0.0001). We divided the patients into four groups, in relation to the results of the tests for specific IgG and C. trachomatis isolation. Seven out of 10 had positive isolation and negative IFA, 5 out of 8 had positive isolation and negative IFA. Twenty five out of 28 pregnant women had negative isolation and positive IFA, finally, 63 out of 76 had both tests negative. Statistical analysis using the McNemar proportion-comparison test suggests that IgG's presence is highly significant in pregnant women with respect to other groups (p < 0.001). Our results suggest that the demonstration of IgG is not enough for diagnostic purposes, except in infertile women with a previous history of infection with C. trachomatis. We isolated C. trachomatis in 20% of the newborns tested and 10% were also positive for IgM IFA. The diagnosis was improved by combining both techniques. These results show the importance of the detection of C. trachomatis in youngsters to avoid infertility and in pregnant women to prevent newborn infections and the possibility of premature births and low weight babies.

Inmuniead y Caldida albicans: un estudio de poblaciones y subpoblaciones linfocitarias / Inmmunity and Candida albicans: a study of lymphocytari popolations and subpopulations

En este trabajo se presentan los resultados de un estudio inmunológico, realizado en 13 pacientes con síndrome de hipersensibilidad a la Candida. Los estudios micológicos confirmaron los hallazgos clínicos de candidiasis. En cada uno de los casos, se realizó cultivo, tipificación y recuente de colonias, que fluctuaron entre 9 800 y 90 000 unidades formadores de colonias de C. albicans/g de materia fecal peso húmedad. Las pruebas de hipersensibilidad cutánea fueron realizadas con 4 antígenos. Todos los pacientes mostraron positividad en la lectura precoz y negatividad en la tardía, a excepción de 4 de ellos. La inmunogluvina E fue cuantificada por ELISA y se encontraron valores por encima de lo normal en 84%de los casos. Los valores hallados fluctuaron entre 31 y 1 000 Ul/ml. La cantidad de LB fue normal en todos los casos, mientras que el de Lt fue normal en 46%y disminuido en 54%de los pacientes. En ambas determinaciones se usó el método de las rosetas. En subpoblaciones de LT se determinaron por técnica de inmunofluorescencia indirecta, con anticuerpos monoclonales. Los CD4+ (cooperadores/inductores) se encontraron disminuidos en 77%de los casos mientras que los CDB+ (citotómicos/supresores) estuvieron normales en la mayoría de ellos (84%). De acuerdo con estos resultados se puede deducir que existe una estrecha relación entre candidiasis, IgE aumentada e inmunodeficiencia fundamentalmente de la inmunidad mediada por células y en especial de las subpoblaciones CD4+. Se propone que en el momento de programar el esquema terapéutico, se incluya un tratamiento inmunomodelador, si fuera necesario, con la finalidad de lograr mayor eficacia terapéutica.

Inmunidad y Candida albicans: un estudio de poblaciones y subpoblaciones linfocitarias / Immunity and Candida albicans: a population and linfocitary sub-population study

En este trabajo se presentan los resultados obtenidos en un estudio inmunológico, realizado en un grupo de 13 pacientes con síndrome de hipersensibilidad a la Candida. Los estudios micológicos confirmaron los hallazgos clínicos de candidiasis. En cada uno de los casos se realizó cultivo, tipificación y recuento de colonias, que oscilaron entre 9.800 y 90.000 unidades formadoras de colonias de C. albicans/g de materia fecal peso húmedo. Las pruebas de hipersensibilidad cutánea fueron realizadas con 4 antígenos. Todos los pacientes mostraron positividad en la lectura precoz y negatividad en la tardía a excepción de 4 de ellos. La inmunoglobulina E fue cuantificada por ELISA y se encontraron valores por encima de lo normal en el 84% de los casos. Los valores hallados oscilaron entre 31 y 1000 Ul/ml. El número de LB fue normal en todos los casos mientras que el de LT fue normal en el 46% y disminuídos en el 54% de los pacientes. En ambas determinaciones se usó el método de las rosetas. Las subpoblaciones de LT se determinaron por técnica de inmunofluorescencia indirecta, con anticuerpos monoclonales. Los CD4+ (cooperadores/inductores) se encontraron disminuídos en el 77% de los casos mientras que los CD8+ (citotóxicos/supersores) estuvieron normales en la mayoría de ellos (84%). De acuerdo a estos resultados se puede deducir que existe una estrecha relación entre candidiasis, IgE aumentada e inmunodeficiencia fundamentalmente de la inmunidad mediada por células y en especial de las ...