Immune cells transferred by colostrum do not influence the immune responses to foot-and-mouth disease primary vaccination.

Little is known about the influence of maternal antibodies and immune cells transferred through colostrum on the immune responses of calves to the currently used foot-and-mouth disease (FMD) vaccines. Here we evaluated the humoral and cellular immune responses induced by vaccination of colostrum-deprived calves and calves that received equivalent amounts of colostrum preparations that differed in the presence or absence of maternal immune cells but contained the same quantity and quality of anti-foot-and-mouth disease virus (FMDV) antibodies. Three groups of 32-d-old calves (n = 3 per group) were deprived of colostrum and fed either whole immune colostrum or a cell-free colostrum preparation containing only anti-FMDV antibodies. All groups were immunized with 1 dose of an oil-adjuvanted commercial vaccine. Blood samples were collected periodically before vaccination and weekly after vaccination. Immune responses specific to FMDV were assessed based on T-cell proliferation, IFN-γ production, total and neutralizing serum antibodies, and isotype profile. All vaccinated calves developed IFN-γ and lymphoproliferative responses, irrespective of the colostrum received. Colostrum-deprived animals responded to vaccination with a primary IgM response followed by an increase of IgG1 titers. Conversely, antibody titers decreased in all colostrum-fed calves after vaccination. This study demonstrates for the first time that maternal immune cells transferred to the calves through colostrum do not modify immune responses to FMD vaccine, and it confirms the interference of maternal antibodies in the induction of humoral but not cell-mediated immune responses.

Comparative histopathological and immunological study of two field strains of chicken anemia virus.

Infection of poultry with chicken anemia virus (CAV) is implicated in several field problems in broiler flocks due to the immunosuppression generated and, consequently, the increased susceptibility to secondary infections. Recently, we have reported an increased occurrence of clinical cases caused by CAV strains distantly related to those commonly used for vaccination. In order to understand the behavior of two Argentinean CAV strains (CAV-10 and CAV-18) in two-week-old chickens, an immune and histopathological study was performed. Neither mortality nor clinical signs were observed in the infected or control groups. Thymus lobes from chickens infected with both CAV viruses were smaller compared to the negative control group. At 14 days post-infection (dpi), only chickens inoculated with CAV-10 show a severe depletion of lymphocytes in the thymus cortex and in follicles from the bursa of Fabricius. Also thymopoiesis disorders, such as reduction in the percentage of total DP (CD4 + CD8α+) thymocytes and alteration in the percentages of DP subpopulations, were more important in animals inoculated with the CAV-10 than the CAV-18 strain. In addition, only animals infected with CAV-10 show a decrease in CD8αß splenocytes. Altogether our results show that, although both Argentinean CAV strains produce subclinical infections in chickens causing immunosuppression at 14 dpi, they might differ in their in vivo pathogenicity.

The role of viral particle integrity in the serological assessment of foot-and-mouth disease virus vaccine-induced immunity in swine.

The efficacy of foot-and-mouth disease virus (FMDV) inactivated vaccines is mainly dependent on the integrity of the whole (146S) viral particles. If the intact capsids disassemble to 12S subunits, antibodies against internal-not protective epitopes, may be induced. Serological correlates with protection may be hampered if antibodies against internal epitopes are measured. Here we compared the performance of different ELISAs with the virus-neutralization test (VNT) that measures antibodies against exposed epitopes. Sera from pigs immunized with one dose of an expired commercial FMDV vaccine were used. This vaccine contained about 50% of O1/Campos and over 90% of A24/Cruzeiro strains total antigen as whole 146S particles. Specific-total antibodies were measured with the standard liquid-phase blocking ELISA (LPBE). We also developed an indirect ELISA (IE) using sucrose gradient purified 146S particles as capture antigen to titrate total antibodies, IgM, IgG1 and IgG2. A good correlation was found between VNT titers and IgG-ELISAs for A24/Cruzeiro, with the lowest correlation coefficient estimated for IgG2 titers. For O1/Campos, however, the presence of antibodies against epitopes different from those of the whole capsid, elicited by the presence of 12S particles in the vaccine, hampered the correlation between LPBE and VNT, which was improved by using purified O1/Campos 146S-particles for the liquid-phase of the LPBE. Interestingly, 146S particles but not 12S were efficiently bound to the ELISA plates, confirming the efficiency of the IE to detect antibodies against exposed epitopes. Our results indicate that any serological test assessing total antibodies or IgG1 against epitopes exposed in intact 146S-particles correlate with the levels of serum neutralizing antibodies in vaccinated pigs, and might potentially replace the VNT, upon validation. We recommend that antigen used for serological assays aimed to measure protective antibodies against FMDV should be controlled to ensure the preservation of 146S viral particles.

FMD vaccine matching: Inter laboratory study for improved understanding of r1 values.

Foot-and-mouth disease virus (FMDV) is a highly variable RNA virus existing as seven different serotypes. The antigenic variability between and within serotypes can limit the cross-reactivity and therefore the in vivo cross-protection of vaccines. Selection of appropriate vaccine strains is crucial in the control of FMD. Determination of indirect relationships (r1-value) between potential vaccine strains and field strains based on antibody responses against both are routinely used for vaccine matching purposes. Aiming at the investigation of the repeatability, reproducibility and comparability of r1-value determination within and between laboratories and serological tests, a small scale vaccine matching ring test for FMDV serotype A was organized. Well-characterized serum pools from cattle vaccinated with a monovalent A24/Cruzeiro/Brazil/55 (A24) FMD vaccine with known in vivo protection status (homologous and heterologous) were distributed to four laboratories to determine r1-values for the heterologous FMD strains A81/Argentina/87, A/Argentina/2000 and A/Argentina/2001 using the virus neutralization tests (VNT) and liquid phase blocking ELISA (LPBE). Within laboratories, the repeatability of r1-value determination was high for both antibody assays. VNT resulted in reproducible and comparable r1-values between laboratories, indicative of a lack of antigenic relatedness between the A24 strain and the heterologous strains tested in this work, thus corresponding to some of the in vivo findings with these strains. Using LPBE, similar trends in r1-values were observed in all laboratories, but the overall reproducibility was lower than with VNT. Inconsistencies between laboratories may at least in part be attributed to differences in LPBE protocols as well as the in preexisting information generated in each laboratory (such as antibody titer-protection correlation curves). To gain more insight in the LPBE-derived r1-values standard bovine control sera were included in the antibody assays performed in each laboratory and a standardization exercise was performed.

Systemic antibodies administered by passive immunization prevent generalization of the infection by foot-and-mouth disease virus in cattle after oronasal challenge.

The role of passively transferred sera in the protection against aerogenous foot-and-mouth disease (FMD) virus infection in cattle was evaluated using vaccine-induced immune serum preparations obtained at 7 and 26 days post-vaccination (dpv). We showed that circulating antibodies were sufficient to prevent disease generalization after oronasal infection in animals passively transferred with 26-dpv serum but not with the 7-dpv serum. Conversely, conventional FMD vaccination provided clinical protection at 7 dpv, promoting fast and robust antibody responses upon challenge and even though antibody titers were similar to those found in animals passively immunized with 7-dpv serum. These results demonstrate that presence of antigen-specific antibodies is critical to prevent the dissemination of the virus within the animal. Conventional FMD vaccination additionally promoted the deployment of rapid, high titer and isotype-switched antibody responses at systemic and mucosal levels after infection, thus conferring protection even in the presence of low pre-challenge antibody titers.

Foot-and-mouth disease vaccination induces cross-reactive IFN-γ responses in cattle that are dependent on the integrity of the 140S particles.

Interferon-γ (IFN-γ) recall responses against foot-and-mouth disease virus (FMDV) in FMD vaccinated cattle are utilized to study T-lymphocyte immunity against this virus. Here, a recall IFN-γ assay based on a commercial ELISA was set up using 308 samples from naïve and vaccinated cattle. The assay was used to study cross-reactive responses between different FMDV vaccine strains. Blood samples from cattle immunized with monovalent vaccines containing A24/Cruzeiro/Brazil/55, A/Argentina/2001 or O1/Campos/Brazil/58 strains were tested using purified-inactivated FMDV from homologous and heterologous strains. A24/Cruzeiro was the most efficient IFN-γ inducer in all vaccinated animals, both when included in the vaccine or as stimulating antigen. We demonstrate that this was mainly due to the structural stability of the whole viral particle. These results show that IFN-γ production relies on the presence of 140S particles that can maintain their integrity along the incubation process in vitro, and throughout the vaccine's shelf-life, when used in vivo.

Influence of antibodies transferred by colostrum in the immune responses of calves to current foot-and-mouth disease vaccines.

Immunity to currently used oil-adjuvanted inactivated vaccines against foot-and-mouth disease virus (FMDV) has been studied in detail in adult animals; however, the influence of maternally derived antibodies transferred through colostrum (Mat-Abs) in the immune responses of vaccinated calves is less clear. Here, we report the anti-FMDV humoral responses elicited in calves with or without Mat-Abs that received one or two doses of the current tetravalent oil-adjuvanted commercial vaccine used in Argentina. Anti-FMDV (O1/Campos strain) antibodies (Abs) were evaluated by Liquid Phase Blocking ELISA (LPB-ELISA), virus neutralization test (VNT), isotype ELISA (IgG1, IgG2 and IgM) and avidity ELISA, to allow for the first time a more detailed description of the humoral responses elicited. Our results show that primary IgM responses to FMDV vaccination only became evident as Mat-Abs titers decreased. Likewise, prime and boost vaccination schedules, applied 35 days apart to groups of calves with high or low levels of Mat-Abs, showed that the levels of preexisting neutralizing Mat-Abs prevented the loss of total Abs measured by LPB-ELISA but negatively interfered with the induction of virus neutralizing responses. Altogether, these findings indicate that comprehensive serological characterization of immune responses generated after vaccination in calves may reveal important information on the actual effectiveness of vaccination strategies for young animals, particularly in endemic settings.

Targeting antigens to an invariant epitope of the MHC Class II DR molecule potentiates the immune response to subunit vaccines.

Recombinant subunit and peptidic vaccines in general present a reduced immunogenicity in vaccinated individuals with respect to the whole pathogen from which they derived. The generation of strong immune responses to these vaccines requires the use of potent adjuvants, high antigen doses and repetitive vaccinations. In this report, we document the enhanced antibody response obtained against two recombinant subunit vaccines by means of targeting to antigen-presenting cells by a recombinant single chain antibody. This antibody, named APCH1, recognizes an epitope of MHC Class II DR molecule preserved in different animal species, including humans. We showed that vaccinal antigens translationally fused to APCH1 antibody and produced by recombinant baculoviruses in insect larvae (Trichoplusia ni), elicited an increased antibody response in comparison with the same antigens alone or fused to a carrier molecule. These results suggest that targeting of antigens to this invariant MHC Class II epitope has immunopotentiating effects that could circumvent the reduced potency of peptidic or subunit vaccines, opening the possibility of widespread application of APCH1 as a new adjuvant antibody of general use.

A DNA vaccine encoding foot-and-mouth disease virus B and T-cell epitopes targeted to class II swine leukocyte antigens protects pigs against viral challenge.

Development of efficient and safer vaccines against foot-and-mouth disease virus (FMDV) is a must. Previous results obtained in our laboratory have demonstrated that DNA vaccines encoding B and T cell epitopes from type C FMDV, efficiently controlled virus replication in mice, while they did not protect against FMDV challenge in pigs, one of the FMDV natural hosts. The main finding of this work is the ability to improve the protection afforded in swine using a new DNA-vaccine prototype (pCMV-APCH1BTT), encoding FMDV B and T-cell epitopes fused to the single-chain variable fragment of the 1F12 mouse monoclonal antibody that recognizes Class-II Swine Leukocyte antigens. Half of the DNA-immunized pigs were fully protected upon viral challenge, while the remaining animals were partially protected, showing a delayed, shorter and milder disease than control pigs. Full protection in a given vaccinated-pig correlated with the induction of specific IFNγ-secreting T-cells, detectable prior to FMDV-challenge, together with a rapid development of neutralizing antibodies after viral challenge, pointing towards the relevance that both arms of the immune response can play in protection. Our results open new avenues for developing future FMDV subunit vaccines.

Dynamics and predictive potential of antibodies against insect-derived recombinant Leishmania infantum proteins during chemotherapy of naturally infected dogs.

A predictive marker for the success treatment of canine leishmaniasis is required for the application of a more rational therapy protocol, which must improve the probability of cure and reduce Leishmania resistance to drugs. We investigated the dynamics and predictive value of antibodies against insect-derived recombinant L. infantum proteins rKMPII and rTRYP by using an enzyme-linked immunosorbent assay with retrospective serum samples from 36 dogs during treatment of canine leishmaniasis. In the entire group of dogs, concentrations of antibodies against rKMPII and rTRYP significantly decreased earlier than concentrations of antibodies against crude total Leishmania antigen (one versus six months), which suggested that the dynamics of antibodies against recombinant proteins may be useful for assessing clinical improvement after treatment. Interestingly, decreases in antibody concentrations against rKMPII occurred earlier in disease-free dogs than in dogs that remain clinically ill one year after beginning of treatment, which suggested that these antibodies may be useful for predicting disease-free survival one year after the beginning of therapy against canine leishmaniasis.

Seroreactivity against raw insect-derived recombinant KMPII, TRYP, and LACK Leishmania infantum proteins in infected dogs.

The recombinant proteins KMPII, TRYP, and LACK of Leishmania infantum were produced in baculovirus-infected Trichoplusia ni larvae and used to analyze the seroreactivity of 165 dog serum samples by the multiple-well ELISA technique (57 infected dogs with clinical signs, 46 naturally infected and 11 experimentally infected; and 108 non-infected dogs, 76 from non-endemic areas and 32 from endemic areas). Recombinant (r) KMPII was the most recognized antigen, as the majority of infected dogs seroreacted against it (0.75). This is the first report of seroreactivity against rTRYP (0.51) and rLACK (0.42) in L. infantum-infected dogs, since previous studies using recombinant TRYP and LACK proteins produced in prokaryotic systems failed to detect specific seroreactivity. All non-infected dogs were negative for rTRYP and rLACK, and only one of the 32 from endemic areas seroreacted against rKMPII. The results demonstrate that L. infantum-infected dogs develop humoral immunity against rKMPII, rTRYP, and rLACK antigens. There was substantial agreement between crude total L. infantum antigen (CTLA)-based ELISA and rKMPII ELISA (kappa=0.664), although this was higher than that found between the CTLA-based ELISA and rTRYP (kappa=0.427) or rLACK (kappa=0.343) ELISA, which can be interpreted as fair and moderate agreement, respectively. Ninety-three percent of the infected dogs analyzed developed specific antibodies against at least one of these three recombinant antigens. When the three recombinant antigen-based ELISA techniques were evaluated in parallel, almost perfect agreement (kappa=0.880) with CTLA-based ELISA was observed, with a specificity of 0.97 and a sensitivity of 0.93 in relation to CTLA-based ELISA. Further studies using purified recombinant antigens in a single-well test or individually, depending on the objective of the study, are warranted.