Cristina Seki

Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina

Specific serum activity levels against four reference strains of foot-and-mouth disease virus (FMDV) were evaluated from 1634 animals vaccinated with commercial quadrivalent oil vaccines and from 746 unvaccinated, naive animals, using the liquid-phase blocking sandwich ELISA (lpELISA) test. Cows from the FMDV-free area of Argentina were tested for the absence of specific FMDV antibodies (sp FMDV Abs) and those showing lpELISA titres < 1.0 were grouped in lots of 16 animals. They were vaccinated and challenged at 90 days postvaccination (DPV) with one of four virus strains used for vaccine production and control (prototype strains). Serum samples from vaccinated and control cattle were collected 60 and 90 DPV and the level of sp FMDV Abs was determined by lpELISA. Animals were examined for clinical signs of disease. Results show that serum lpELISA titre levels directly correlate with the percentage of protected animals. It was seen that 100, 98, 93 and 87% of the vaccinated cattle with antibody titre levels > or = 2.1 were protected against challenge with serotypes C85, A87,01 Cas and A79, respectively. Evidence is also presented of seroconversion in a sample of 3-5-month-old calves vaccinated in the field, showing lpELISA titres compatible with protection against the four vaccine viruses as long as 150 DPV. Results reported in this paper strongly support the use of the lpELISA test for a rapid and reliable evaluation of the efficacy of FMDV commercial vaccines as well as for the assessment of the immunological status of cattle in FMDV-free and enzootic regions of South America.

Updating of the correlation between lpELISA titers and protection from virus challenge for the assessment of the potency of polyvalent aphtovirus vaccines in Argentina

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.

Assessment of foot and mouth disease vaccine potency by liquid-phase blocking ELISA: a proposal for an alternative to the challenge procedure in Argentina

The lowest expected protection (LEP) at a 95% confidence of 245 foot and mouth disease (FMD) commercial vaccines was calculated from the titres of liquid-phase blocking sandwich ELISA (lpELISA) of cattle sera obtained from 3920 animals at 60 days post-vaccination (d.p.v.) and challenged with live virus at 90 d.p.v. It was found that LEP evaluation is highly specific (i.e. it is able to predict the failure in 100% of the cases) although its ability to predict the challenge (PG test) approval (i.e. sensitivity) comprised only 65% of the vaccines that passed the trial. It was possible, nevertheless, to improve the sensitivity of the evaluation by using an alternative coefficient (Ro), exclusively dependent on the number of animals exhibiting the highest and lowest lpELISA titres in a particular vaccine trial. This coefficient was capable of predicting the PG approval of 90% of the vaccines, yet maintaining acceptable levels of safety (87% of specificity). Based on these results and as a first step towards the replacement of the challenge protocol in Argentina, we propose a swift approval for commercialization of FMD vaccines which are able to reach the highly restricting LEP passmark of 82%, and the rejection of those not reaching the 50% LEP limit. More extensive experience with this new protocol will allow a finer adjustment of the LEP and Ro values and to set more precisely the cut-off points for direct approval or disapproval of vaccines by lpELISA, eliminating the use of live FMDV in the field.

Total and isotype humoral responses in cattle vaccinated with foot and mouth disease virus (FMDV) immunogen produced either in bovine tongue tissue or in BHK-21 cell suspension cultures

The anti-foot and mouth disease virus (FMDV) serum antibody activity of protected and non protected animals immunized with inactivated FMDV originated in either bovine tongue tissue (BTTV vaccines) or BHK-21 cell suspension cultures (BHKV vaccines) was evaluated. The results show that 80-100% of the BTTV immunized and only 40-60% of the BHKV immunized animals with liquid-phase blocking sandwich ELISA (lp ELISA) serum titres of 1.5-1.7 U, were protected against the challenge with any of the four infectious FMDV argentine reference strains. This difference becomes almost marginal among BTTV and BHKV vaccinated animals with a strong anti-FMDV humoral response (i.e. lp ELISA titres > or = 1.95 U). Isotyping of the anti-FMDV response in immunized cattle with low lp ELISA titres revealed that BTTV vaccines were able to induce remarkably higher anti-FMDV IgG1 titres than their BHKV counterparts (i.e. mean titres of 1.95 and 1.35 U. respectively). This difference in specific IgG1 serum levels induced by BTTV and BHKV vaccines seems to be also limited to those animals with low anti-FMDV lp ELISA titres. These results together with the fact that the specific serum IgG1, but not the IgG2, isotype response of 219 vaccinated animals correlates almost linearly with their capacity to pass the challenge, suggests that the superior performance of BTTV vaccines is close related to their ability to raise a stronger anti-FMDV IgG1 response than BHKV vaccines.

Confidence in indirect assessment of foot-and-mouth disease vaccine potency and vaccine matching carried out by liquid phase ELISA and virus neutralization tests.

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.

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies.

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.

Characterization of foot-and-mouth disease virus from outbreaks in Ecuador during 2009-2010 and cross-protection studies with the vaccine strain in use in the region.

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.

Characterization of a type O foot-and-mouth disease virus re-emerging in the year 2011 in free areas of the Southern Cone of South America and cross-protection studies with the vaccine strain in use in the region.

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.

Quantitative single serum-dilution liquid phase competitive blocking ELISA for the assessment of herd immunity and expected protection against foot-and-mouth disease virus in vaccinated cattle.

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.

Development and validation of an ELISA for quantitation of Bovine Viral Diarrhea Virus antigen in the critical stages of vaccine production

Bovine Viral Diarrhea Virus (BVDV) is the causative agent of a worldwide disease. The virus infects bovines of all ages, causing reproductive problems and contaminating biological products of high commercial value. The large-scale production of BVDV vaccines presents the challenge of processing antigenic proteins that are highly susceptible to the processing environment. Potency testing requires the immunization of cattle in order to determine the neutralizing antibodies titers induced by the vaccine. An alternative to the in vivo test is an in vitro measurement of key viral antigens. This paper describes the development and validation of a sandwich-type indirect ELISA that is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV. Validation parameters such as repeatability, intermediate precision, and reproducibility indicated that the developed ELISA constitutes an advanced tool for evaluating the BVDV antigen throughout manufacturing and vaccine release testing.