RESUMEN

Molecular knowledge of virus-antibody interactions is essential for the development of better vaccines and for a timely assessment of the spread and severity of epidemics. For foot-and-mouth disease virus (FMDV) research, in particular, computational methods for antigen-antibody (Ag-Ab) interaction, and cross-antigenicity characterization and prediction are critical to design engineered vaccines with robust, long-lasting, and wider response against different strains. We integrated existing structural modeling and prediction algorithms to study the surface properties of FMDV Ags and Abs and their interaction. First, we explored four modeling and two Ag-Ab docking methods and implemented a computational pipeline based on a reference Ag-Ab structure for FMDV of serotype C, to be used as a source protocol for the study of unknown interaction pairs of Ag-Ab. Next, we obtained the variable region sequence of two monoclonal IgM and IgG antibodies that recognize and neutralize antigenic site A (AgSA) epitopes from South America serotype A FMDV and developed two peptide ELISAs for their fine epitope mapping. Then, we applied the previous Ag-Ab molecular structure modeling and docking protocol further scored by functional peptide ELISA data. This work highlights a possible different behavior in the immune response of IgG and IgM Ab isotypes. The present method yielded reliable Ab models with differential paratopes and Ag interaction topologies in concordance with their isotype classes. Moreover, it demonstrates the applicability of computational prediction techniques to the interaction phenomena between the FMDV immunodominant AgSA and Abs, and points out their potential utility as a metric for virus-related, massive Ab repertoire analysis or as a starting point for recombinant vaccine design.

RESUMEN

Molecular, antigenic and vaccine matching studies, including protective response in vivo, were conducted with a foot-and-mouth disease type O virus isolated during the outbreak in September 2011 in San Pedro, Paraguay, country internationally recognized as free with vaccination in 1997. The phylogenetic tree derived from complete VP(1) sequences as well as monoclonal antibody profiling indicated that this isolate was related to viruses responsible for previous emergencies in free areas of the Southern Cone of South America occurring sporadically between the years 2000 and 2006. Marked differences with the vaccine strain O(1)/Campos, including the loss of reactivity with neutralizing MAbs, were recognized. Levels of protective antibodies induced by the vaccine containing the O(1)/Campos strain against the San Pedro virus and the virus responsible for the previous emergency in 2006 in the Southern Cone assessed by in vitro vaccine matching studies pointed to an insufficient protective response 30 days after vaccination (DPV), which was properly attained at 79 DPV or after revaccination. In agreement with the in vitro assessment, the in vivo challenge in the Protection against Podal Generalization test in cattle indicated appropriate protection for the San Pedro strain at 79 DPV or after revaccination. The complementary conclusions that can be derived from vaccine matching tests designed differently to fit the various objectives intended: prophylaxis, emergency vaccination or incorporation of new field strains into antigen banks, is evaluated. This is the first report of the antigenic and immunogenic characterization of the variants responsible for emergencies in the Southern Cone of South America and the putative impact of the changes on the cross protection conferred by the vaccine strain.

Asunto(s)

RESUMEN

During the years 2009 and 2010 relevant epidemic waves of foot-and-mouth disease (FMD) serotype O occurred in Ecuador, representing a great drawback for the last stages of the ongoing eradication program in South America. This study describes the molecular and antigenic characterizations of 29 isolates collected from various regions in the country and their relationship to the vaccine strain. The phylogenetic tree derived from sequences spanning the complete VP(1) protein showed that, despite the widespread origin of the viruses, they were all related among themselves and to previous isolates occurring in 2008, with around 10% difference with the vaccine strain O1/Campos. The high level of sequence conservation among different isolates in the various regions of Ecuador pointed to a common origin, suggesting animal movements as possible sources of viral spread. Monoclonal antibody profiling grouped the isolates in two major reactivity patterns which differed from that of the vaccine strain. Both profiles showed loss of reactivity with the same four MAbs, three of them with neutralizing properties. Additional sites were lost in the profile representing most of the 2010s viral samples. Levels of protective antibodies induced by the vaccine against the field strains assessed by in vitro vaccine matching studies also pointed to an increased temporal pattern of loss of a protective response. Moreover, results obtained with in vivo challenge in the protection against podal generalization test in cattle, clearly indicated lack of appropriate protection of the Ecuadorian field strains by the vaccine virus in use, which in the case of a 2010 variant was observed even after revaccination.

Asunto(s)

RESUMEN

The necessity of avoiding the use of animals in vaccine potency testing has been widely recognized. The repeatability and reproducibility of the Expected Percentage of Protection (EPP) as a serological potency surrogate for A24 Cruzeiro foot-and-mouth disease virus (FMDV) strain was assessed, and compared with the results obtained with challenge in the Protection against Podal Generalization (PPG) test. To determine the EPPs, the serum titers obtained by liquid phase blocking competitive ELISA (lpELISA) and virus neutralization (VNT) in 10 potency trials using the same A24 Cruzeiro vaccine, were interpolated into previously validated logit transformation curves that correlate PPG with serology. Indirect serological assessment of vaccine matching between the serotype A FMDV strains A24 Cruzeiro and A/Argentina/01 was also carried out by lpELISA and VNT. The results obtained in this study strongly support the replacement of challenge tests for vaccine potency by indirect serological assays, at least for A24 Cruzeiro FMDV strain. While determination of EPPs by lpELISA titers showed an excellent repeatability, reproducibility and concordance with PPG for vaccine potency, assessments of cross-protection by VNT titers were more consistent with the PPG outcome.

Asunto(s)

RESUMEN

A single serum-dilution liquid phase ELISA (slpELISA) was standardized to be used for serological evaluation of herd immunity against foot-and-mouth disease. The absorbance value at a dilution 1:64 of each serum sample was interpolated in a standard curve by plotting the antibody titers of six control sera determined by end point dilution liquid phase ELISA (lpELISA), against the absorbance values for the same control sera at 1:64 dilutions. A straight line was obtained by linear regression analysis (r>0.90) in the titer range of 1.40-2.40. The reliability of the antibody titers was confirmed by the simultaneous titration of 60 cattle sera by slpELISA and lpELISA, which showed an acceptable correlation (R(2)>0.87) for viral strains A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial. Titers obtained by both methods were not significantly different (p>0.05), thus confirming that slpELISA could be used successfully to replace the conventional serial dilution ELISA for the assessment of protection status of cattle in epidemiological studies. In addition, this quantitative slpELISA provides an adequate method for monitoring the effectiveness of vaccination campaigns and is also suitable for the assessment of seroconversion of naive animals during early stages of infection.

Asunto(s)

RESUMEN

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.

Asunto(s)

RESUMEN

Routine vaccination campaigns are carried out in Argentina twice a year, involving more than 100 million doses of foot-and-mouth disease (FMD) vaccine. Although the challenge test in cattle has not been totally replaced for the assessment of FMD vaccine potency, Argentine Animal Health authorities have used an indirect alternative method based on specific correlation studies of protection against podal generalization (PPG) tests performed in cattle with a validated liquid phase blocking ELISA (lpELISA). The change of vaccine formulations that took place after the 2000-2001 outbreaks, generated a gap in the correlation between lpELISA titers and PPG for the new FMD virus strains. A reappraisal of the correlation between lpELISA titers measured at 60 dpv and virus challenge by the PPG method at 90 dpv, performed for the four virus strains presently included in the Argentine vaccine is presented in this work. The data were obtained from 40 bovine challenge trials (647 sera) performed using exclusive batches of commercial vaccine from the year 2001 to January 2008 for A24/Cruzeiro, A/Argentina/2001, O1/Campos and C3/Indaial FMD virus strains. Curves of percentage of expected protection (EPP) versus lpELISA titers were obtained by logit regression for A/Argentina/2001, O1/Campos and C3/Indaial strains, but not for A24/Cruzeiro strain. The concordance between the direct and indirect tests using an EPP cut off value of 75% (82%, kappa = 0.62), in agreement with data originating from many years of vaccine control in Argentina, remarks the relevance of the acceptance of indirect alternatives to in vivo potency testing.

Asunto(s)

RESUMEN

The successful sanitary campaign implemented to control the 2000-2002 outbreaks of foot-and-mouth disease virus (FMDV) in Argentina was greatly assisted by the combination of an ELISA test (3ABC-ELISA) that detects antibodies directed against FMDV viral non-structural proteins (NSPs) and a liquid phase blocking competitive ELISA (lpELISA) for the detection of antibodies against the viral structural proteins (SPs). The combined use of these two assays in large-scale analysis of field samples allowed for a clear differentiation between infected and uninfected animals, with high specificity and sensitivity, regardless of the animal's vaccination status. In order to validate the application in indirect vaccine potency assays and assessment of vaccination efficiency, a preliminary correlation between serological response and protection from challenge with O1/Campos and A/Arg/01 FMD virus strains was established with data derived from commercial vaccine series challenge trials. Determination of antibodies to NSPs in vaccinated and revaccinated animals proved helpful in the analysis of vaccine purity. A review and discussion of the epidemiological status of cattle herds and real time monitoring of FMD in Argentina using these assays before, during and after the outbreaks is presented.

Asunto(s)

RESUMEN

This paper describes the antigenic and molecular characterisation of foot-and-mouth disease virus (FMDV) strains isolated during the 2000-2002 epidemic in Argentina, and the strategy implemented for disease control. Two different FMDV serotypes, O and A, were involved. Of the various field isolates studied, two distinct O1 lineages (strains Corrientes/00 and Misiones/00) and two serotype A lineages (A/Argentina/00 and A/Argentina/01 prototypes) were identified. The genome sequences of these strains were compared with sequences of previous regional isolates and sequences of vaccine strains. O1 strains were found to be related to regional strains while serotype A strains were found to be more distanced from them. The updating of the antigenic composition of the vaccines used in the emergency was a key issue, since the outbreaks stopped shortly after the implementation of the vaccination programs. The O1 strains quickly disappeared from the field following strict control measures and the use of vaccines containing O1/Campos strain. However, in the case of the A serotype strains, the situation was different, since the use of a vaccine containing strain A24/Cruzeiro yielded acceptable levels of protection only after re-vaccination. Therefore, the new field strains A/Argentina/00 and A/Argentina/01 were incorporated into the vaccine, leading to an effective control of the disease. Viral circulation greatly diminished, as indicated by the significant reduction in the number of outbreaks and in the number of animals with antibodies against non-structural proteins. Satisfactory levels of protective antibodies were subsequently detected in the cattle population (above 75% protection). The absence of outbreaks after January 2002 indicated that the epidemic was controlled.

Asunto(s)

Asunto(s)

Asunto(s)