Humoral and T cell-mediated immune responses to bivalent killed bovine viral diarrhea virus vaccine in beef cattle.

The objective of this research project was to evaluate the antibody and cell-mediated immune responses to a multivalent vaccine containing killed bovine viral diarrhea virus (BVDV) types 1 and 2. Twenty castrated male crossbred beef cattle (350-420kg body weight) seronegative to BVDV were randomly divided into two groups of 10 each. Group 1 served as negative mock-vaccinated control. Group 2 was vaccinated subcutaneously twice, 3 weeks apart, with modified live bovine herpesvirus 1, parainfluenza 3 virus and bovine respiratory syncytial virus diluted in diluent containing killed BVDV type 1 (strain 5960) and type 2 (strain 53637) in an adjuvant containing Quil A, Amphigen, and cholesterol. Serum samples were collected from all cattle at days -21, 0, and days 21, 28, 35, 56 and 70 post-vaccination. Standard serum virus neutralization tests were performed with BVDV type 1 (strain 5960) and type 2 (strain 125C). Anticoagulated blood samples were collected at day 0, and days 28, 35, 56 and 70 post-vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated, stimulated with live BVDV type 1 (strain TGAN) and type 2 (strain 890) and cultured in vitro for 4 days. Supernatants of cultured cells were collected and saved for interferon gamma (IFNgamma) indirect enzyme-linked immunosorbent assay (ELISA). Four-color flow cytometry was performed to stain and identify cultured PBMC for three T cell surface markers (CD4, CD8, and gammadelta TCR) and to detect the activation marker CD25 (alpha chain of IL-2 receptor) expression. The net increase in %CD25+ cells (Delta%CD25+) of each T cell subset of individual cattle was calculated. The results of all post-vaccination weeks of each animal were plotted and the areas under the curve of each T cell subset were statistically analyzed and compared between groups. The mean area under the curve of the Delta%CD25+ data for days 0-70 of all subsets, except CD4-CD8+gammadelta TCR- (cytotoxic) T cell subset of both BVDV types 1 and 2 stimulated cells, of the vaccinated group were significantly higher than the control group (P<0.05). IFNgamma production by PBMC from the vaccinated group showed significantly higher results (P<0.05) than the control group in the BVDV types 1 and 2 stimulated cells for at least some time points after vaccination. The vaccinated group also had significantly (P<0.0001) higher neutralizing antibody titers than the control group from day 28 onward.

Comparison of humoral and T-cell-mediated immune responses to a single dose of Bovela® live double deleted BVDV vaccine or to a field BVDV strain.

The objective of this study was to determine and compare the humoral and cellular immune responses of calves exposed to a single dose of Bovela® bovine viral diarrhea virus (BVDV) live double deleted vaccine or a field strain virus (FSV) of BVDV type 2 (strain 890). Thirty seronegative, colostrum-deprived 5 month-old Holstein steer calves that tested negative for persistent BVDV by ear notch immunohistochemistry and seronegative to BVDV types 1 and 2 were used. Calves were screened by multi-parameter flow cytometry (MP-FCM) 1 week before vaccination to ensure that they were negative for T cell responses to the BVDV types 1 and 2 viruses in the Bovela® vaccine. Calves were assigned to 3 treatment groups: control (PBS), FSV inoculated, and Bovela® vaccinated. The humoral response was tested by standard serum virus neutralization (SVN) test to BVDV types 1 (Singer strain) and 2 (strain 125). The response by CD4, CD8, and gamma delta (γδ TCR) T cells was evaluated by MP-FCM using individual BVDV types 1 and 2 from Bovela® vaccine as recall antigens at 5, 6, and 7 weeks after vaccination. Activation markers used were upregulation of surface CD25 (IL-2R), intracellular interferon gamma (IFNγ) and intracellular interleukin 4 (IL-4). Each T cell subset was evaluated for increased expression of each activation marker compared to non-antigen stimulated cells of the same animal. All Bovela® vaccinated and FSV inoculated calves produced SVN antibodies to both BVDV types 1 and 2 while control animals remained seronegative throughout the study. The mean (weeks 5, 6, and 7) T cell recall responses to Bovela® BVDV type 1 and type 2 recall antigens were numerically higher in all three T cell subsets (CD4, CD8, and γδ TCR) for all three activation markers (CD25, IFNγ, and IL-4) when compared to either the control animals or to the FSV inoculated animals. These differences were often, but not always, statistically significant (P<0.05).

Effects of porcine reproductive and respiratory syndrome virus-infected antigen-presenting cells on T cell activation and antiviral cytokine production.

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to suppress T cell expression of CD25 (alpha chain of interleukin [IL]-2 receptor), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) was determined by flow cytometry in naive porcine T cells in response to mitogen (concanavalin A) and cytokine inducers (phorbol 12-myristate 13-acetate plus ionomycin [PMA/I]). Four PRRSV isolates of varying clinical virulence and three different types of porcine myeloid antigen-presenting cells (APCs) were used. T cells cultured with monocytes infected with virulent PRRSV (VR-2385, SDSU-73, and VR-2332), but not with a vaccine strain (Ingelvac PRRS MLV; Boehringer Ingelheim Vetmedica, St. Joseph, MO), demonstrated significantly reduced CD25 expression (%CD25(+)) and IFN-gamma expression (%IFN-gamma (+)) compared with T cells incubated with uninoculated monocyte cultures. T cells cultured with monocytes infected with all four PRRSV isolates demonstrated significantly reduced %TNF-alpha (+). The significant reduction of %CD25(+), %IFN-gamma (+), and %TNF-alpha (+) was not detected in T cells cultured with monocyte-derived macrophages (MDMs) and immature monocyte-derived dendritic cells (MDCs) infected with any PRRSV isolates. Heat-inactivated PRRSV did not induce significantly reduced T cell responses in any APC cultures. The reduction of T cell response in monocyte cultures was not due to PRRSV-induced T cell death. Gene expression of IL-10 detected by semiquantitative reverse transcriptase-polymerase chain reaction was significantly increased in virulent PRRSV-infected monocyte cultures after PMA/I, but not concanavalin A, stimulation compared with IL-10 gene expression from uninoculated monocyte cultures. Increased IL-10 gene expression contributed to significantly reduced %IFN-gamma (+) and %TNF-alpha (+), but not %CD25(+), as determined by IL-10 neutralization assay. This study reports that PRRSV has the ability to suppress T cell responses. The suppressive ability of PRRSV is associated with viral virulence and is mediated by virus-infected monocytes, but not by virus-infected MDMs and immature MDCs.

Immune responses and protection by vaccine and various vaccine adjuvant candidates to virulent porcine reproductive and respiratory syndrome virus.

Various vaccine adjuvant candidates were assessed with the modified-live porcine reproductive and respiratory syndrome virus (MLV PRRSV) (Ingelvac PRRS MLV) vaccine. Their influence on humoral-mediated immune (HMI) and cell-mediated immune (CMI) responses as well as protection from virulent PRRSV challenge (MN-184) was evaluated. Ninety seronegative pigs were randomly divided into nine groups of 10 pigs. One group received MLV vaccine alone. Five groups received MLV vaccine with either bacterial endotoxin-derived adjuvant (ET), mixed open reading frame 5 (ORF5) peptides derived from various PRRSV isolates, porcine interferon alpha (IFNalpha), polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC), or porcine interleukin-12 (IL-12). One group did not receive MLV vaccine but was immunized with ORF5 peptides conjugated with cholera toxin (ORF5 peptide/CT). Two groups served as challenged and unchallenged non-vaccinated controls. Four-color flow cytometry was utilized to simultaneously identify three major porcine T-cell surface markers (CD4, CD8, and gammadelta TCR) and detect activation marker CD25 (alpha chain of IL-2 receptor) or intracellular IFNgamma. The MLV PRRSV vaccine alone successfully primed CD4(-)CD8(+)gammadelta- T-cells as demonstrated by a significant increase in %IFNgamma+ cells when live PRRSV was used as a recall antigen. Booster immunizations of mixed ORF5 peptides and co-administration of IL-12 with MLV PRRSV vaccine significantly enhanced IFNgamma expression by some T-cell subsets (CD4(-)CD8(+)gammadelta+ and CD4(-)CD8(-)gammadelta+ for mixed ORF5 peptides and CD4(+)CD8(+)gammadelta- and CD4(-)CD8(+)gammadelta+ for IL-12). All groups receiving MLV-vaccine with or without adjuvants had reduced lung lesions after challenge. The group immunized with only ORF5 peptide/CT did not have significant T-cell recall responses and was not protected from challenge. Expression of IFNgamma by several T-cell subsets correlated with reduced lung lesions and viremia, whereas expression of CD25 did not. Expression of surface CD25 did not correlate with IFNgamma production. PRRSV ELISA s/p ratio prior to challenge also correlated with reduced lung lesions and viremia. In conclusion, booster immunizations of the mixed ORF5 peptides and co-administration of IL-12 effectively enhanced the CMI response to MLV vaccine. However, neither adjuvant significantly contributed to reducing clinical effects when compared to MLV alone.

Induction of antigen-specific T-cell subset activation to bovine respiratory disease viruses by a modified-live virus vaccine.

OBJECTIVE: To determine the efficacy of a modified-live virus vaccine containing bovine herpes virus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), parainfluenza virus 3, and bovine viral diarrhea virus (BVDV) types 1 and 2 to induce neutralizing antibodies and cell-mediated immunity in naïve cattle and protect against BHV-1 challenge. ANIMALS: 17 calves. PROCEDURES: 8 calves were mock-vaccinated with saline (0.9% NaCl) solution (control calves), and 9 calves were vaccinated at 15 to 16 weeks of age. All calves were challenged with BHV-1 25 weeks after vaccination. Neutralizing antibodies and T-cell responsiveness were tested on the day of vaccination and periodically after vaccination and BHV-1 challenge. Specific T-cell responses were evaluated by comparing CD25 upregulation and intracellular interferon-gamma expression by 5-color flow cytometry. Titration of BHV-1 in nasal secretions was performed daily after challenge. Results-Vaccinated calves seroconverted by week 4 after vaccination. Antigen-specific cell-mediated immune responses, by CD25 expression index, were significantly higher in vaccinated calves than control calves. Compared with control calves, antigen-specific interferon-gamma expression was significantly higher in calves during weeks 4 to 8 after vaccination, declining by week 24. After BHV-1 challenge, both neutralizing antibodies and T-cell responses of vaccinated calves had anamnestic responses to BHV-1. Vaccinated calves shed virus in nasal secretions at significantly lower titers for a shorter period and had significantly lower rectal temperatures than control calves. CONCLUSION AND CLINICAL RELEVANCE: A single dose of vaccine effectively induced humoral and cellular immune responses against BHV-1, BRSV, and BVDV types 1 and 2 and protected calves after BHV-1 challenge for 6 months after vaccination.

Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine.

OBJECTIVE: To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays. ANIMALS: 45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP. PROCEDURES: Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine. RESULTS: Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-gamma had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-gamma was not correlated as well with results for the other tests. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP.

Heterologous challenge in the presence of maternally-derived antibodies results in vaccine-associated enhanced respiratory disease in weaned piglets.

Control of influenza A virus (IAV) in pigs is done by vaccination of females to provide maternally-derived antibodies (MDA) through colostrum. Our aim was to evaluate if MDA interfere with IAV infection, clinical disease, and transmission in non-vaccinated piglets. In the first study, naïve sows were vaccinated with H1N2-δ1 whole inactivated virus (WIV) vaccine. In a follow-up study seropositive sows to 2009 pandemic H1N1 (H1N1pdm09) were boosted with H1N1pdm09 WIV or secondary experimental infection (EXP). MDA-positive pigs were challenged with homologous or heterologous virus, and MDA-negative control groups were included. WIV-MDA piglets were protected from homologous infection. However, piglets with WIV-derived MDA subsequently challenged with heterologous virus developed vaccine associated enhanced respiratory disease (VAERD), regardless of history of natural exposure in the sows. Our data indicates that although high titers of vaccine-derived MDA reduced homologous virus infection, transmission, and disease, MDA alone was sufficient to induce VAERD upon heterologous infection.

T cells from a high proportion of apparently naive cattle can be activated by modified vaccinia virus Ankara (MVA).

Modified vaccinia virus Ankara (MVA) was used as a vector to express genes from bovine respiratory syncytial virus (BRSV). Using these recombinant viruses as recall antigens for cells from BRSV-immuned cattle proved to be problematic because non-recombinant MVA itself frequently stimulated high levels of T lymphocyte activation. This phenomenon was observed in a high percentage of cattle from multiple herds. Gamma delta TCR(+) T cells were more sensitive to activation by MVA than other classes of T cells. A serological assay for MVA neutralization detected low, fluctuating titers of serum virus neutralizing (SVN) activity toward MVA in some cattle, but these were lower titers than those observed in cattle that underwent MVA vaccination. T cell reactivity in non-vaccinated cattle did not correlate significantly (p > 0.05) with SVN activity, undermining the notion that any adaptive immune response was responsible for the observed T cell sensitivity. More probable explanations are that MVA has mitogenic or superantigenic properties, or that the virus induces gammadelta TCR(+) T cell activation through interactions with innate pattern recognition receptors.

Development of a novel method of lytic phage delivery by use of a bacteriophage P22 site-specific recombination system.

Bacteriophage therapy represents a potential alternative to the use of antibiotics to control proliferation of pathogenic bacteria. As an alternative to the strategy where a limited number of doses of large numbers of lytic bacteriophages are administered, a novel method delivery system was developed so that phages are continually released into the culture. Specifically, a non-pathogenic Escherichia coli strain was constructed that was lysogenic for a lytic mutant of bacteriophage lambda. This lysogen was shown to be effective at decreasing the number of lambda-sensitive E. coli in vitro. Construction of this E. coli strain was accomplished by development of a plasmid-based system utilizing the site-specific recombination machinery of bacteriophage P22 to integrate DNA constructs into the host chromosome. This recombination system is useful for strain construction and other genetic manipulations in both E. coli and Salmonella enterica serovars.

Divergent immune responses and disease outcomes in piglets immunized with inactivated and attenuated H3N2 swine influenza vaccines in the presence of maternally-derived antibodies.

Live-attenuated influenza virus (LAIV) prime-boost vaccination previously conferred protection against heterologous H3N2 swine influenza challenge, including in piglets with maternally derived antibodies (MDA). Conversely, a whole-inactivated virus (WIV) vaccine was associated with enhanced disease. This study was aimed at identifying immune correlates of cross-protection. Piglets with and without MDA received intramuscular adjuvanted WIV or intranasal LAIV, and were challenged with heterologous H3N2. WIV induced cross-reactive IgG, inhibited by MDA, and a moderate T cell response. LAIV elicited mucosal antibodies and T cells cross-reactive to the heterologous challenge strain. The presence of MDA at LAIV vaccination blocked lung and nasal antibody production, but did not interfere with T cell priming. Even without mucosal antibodies, MDA-positive LAIV vaccinates were protected, indicating a likely role for T cells. Based on the data, one LAIV dose can induce cell-mediated immunity against antigenically divergent H3N2 influenza virus despite passive antibody interference with humoral immune responses.

Canine peripheral blood lymphocyte phenotyping by 7-color multiparameter flow cytometry.

OBJECTIVE: To characterize baseline canine lymphocyte phenotypes including lymphocytes coexpressing multiple markers by novel 7-color multiparameter flow cytometry. STUDY DESIGN: Fresh canine peripheral blood lymphocytes of 79 healthy 26-week-old Beagle or Beagle-mix dogs were stained and analyzed. RESULTS: The high number of samples and acquired flow data (averaging 1.9 x 10(5) cells/sample) allowed the detection of minor lymphocyte subsets coexpressing multiple lymphocyte markers. The averaged percentages of major lymphocyte subsets of CD3+, CD4+, CD8+, CD21+ and gammadelta TCR+ cells from this study were 74.0, 43.6, 14.3, 9.6, and 0.2, respectively, which were comparable but uniquely different from other reports as they were simultaneously detected in the same sample. We demonstrated that the commonly used CD21 and CD3 monoclonal antibody (mAb) clones, previously recommended not to be used in the same staining, could and should be used together with the proper steps of lymphocyte gating. We found a high percentage (10.3%) of unidentified CD21- CD3+ CD4- CD8-gammadelta TCR- lymphocyte subset that has never been reported. The intensive gating strategy and the mean percentages of each lymphocyte subset to their parent subsets and to the total lymphocyte population are presented and discussed. CONCLUSION: The canine lymphocyte phenotypes were fully characterized. This novel multiparameter flow cytometry method is a powerful approach to in-crease the accuracy of lymphocyte phenotyping in dogs.

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs.

The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1Δ126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1Δ126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1Δ126 TX98 infected pigs were minimal. However, intracellular IFN-γ data indicate that the attenuated virus induced virus-specific CD4(+)CD8(-), CD4(+)CD8(+), CD4(-)CD8(+), and γδ T cells within 28 days. The IFN-γ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4(+)CD8(+) cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replication of the H1N1 challenge virus. Vaccination with NS1Δ126 TX98 was associated with significantly lower levels of Th1-associated cytokines in infected lungs but provided partial cross-protection against the H1N1 challenge. These results demonstrate that NS1Δ SIV vaccines can elicit cell-mediated cross-protection against antigenically divergent strains.

Comparison of humoral and cellular immune responses to inactivated swine influenza virus vaccine in weaned pigs.

Humoral and cellular immune responses to inactivated swine influenza virus (SIV) vaccine were evaluated and compared. Fifty 3-week-old weaned pigs were randomly divided into the non-vaccinated control group and vaccinated group containing 25 pigs each. Pigs were vaccinated intramuscularly twice with adjuvanted UV-inactivated A/SW/MN/02011/08 (MN/08) H1N2 SIV vaccine at 6 and 9 weeks of age. Whole blood samples for multi-parameter flow cytometry (MP-FCM) and serum samples for hemagglutination inhibition (HI) assay were collected at 23 and 28 days after the second vaccination, respectively. A standard HI assay and MP-FCM were performed against UV-inactivated homologous MN/08 and heterologous pandemic A/CA/04/2009 (CA/09) H1N1 viruses. While the HI assay detected humoral responses only to the MN/08 virus, the MP-FCM detected strong cellular responses against the MN/08 virus and significant heterologous responses to the CA/09 virus, especially in the CD4+CD8+ T cell subset. The cellular heterologous responses to UV-inactivated virus by MP-FCM suggested that the assay was sensitive and potentially detected a wider range of antigens than what was detected by the HI assay. Overall, the adjuvanted UV-inactivated A/SW/MN/02011/08 H1N2 SIV vaccine stimulated both humoral and cellular immune responses including the CD4-CD8+ T cell subset.

Cell-mediated immunity evaluation in foals infected with virulent equine herpesvirus-1 by multi-parameter flow cytometry.

The cell-mediated immune (CMI) response of foals to virulent equine herpesvirus-1 (EHV-1) infection was evaluated by multi-parameter flow cytometry (FCM). Ten 7-8-month-old EHV-1 seronegative foals were infected intranasally with virulent EHV-1 and 10 foals served as uninfected controls. Blood samples were collected 6 and 7 weeks after infection to test for specific CMI responses to live heterologous EHV-1 recall antigen. The activation markers included major histocompatibility complex class II (MHC II), intracellular interferon gamma (IFN-gamma) and interleukin 4 (IL-4). The results from both tests were averaged before statistical analysis. Following EHV-1 stimulation, the MHC II expression index (EI) increased significantly in CD2+CD4+CD8- and CD2+CD4-CD8+ subsets of the infected group. At 4 days after incubation, the non-antigen stimulated CD2+CD4-CD8- subset of the infected group expressed a high percentage (61.1%) of MHC II. When stimulated with EHV-1, the MHC II expression declined significantly but remained at a relatively high percentage (34.4%). The IFN-gamma EI was significantly higher in infected foals in all major T cell subsets (CD2+) while only the CD2+CD4+CD8- subset showed a significant increase in intracellular IL-4 EI. The FCM results showed strong specific CMI responses to EHV-1 by all three tested parameters compared to the control group (p<0.01). The high MHC II expression in the CD2+CD4-CD8- subset suggests that this T cell subset may represent a gammadelta TCR repertoire and thereby plays an important role as antigen presenting cells in the horse, as reported in other species. Being able to simultaneously quantify the frequency of specific lymphocyte subsets and the expression of cytokines that characterize activation of lymphocytes and protective CMI by multi-parameter FCM enables evaluation of subset-specific CMI responses to EHV-1 infection. This system can be applied to measure CMI responses to other equine vaccines and pathogens.

Evaluation of the cell-mediated immune response to reduced doses of Mycobacterium avium ssp. paratuberculosis vaccine in cattle.

Vaccination against Johne's disease with an inactivated, oil-adjuvanted Mycobacterium avium ssp. paratuberculosis (MAP) bacterin can reduce clinical signs in infected herds; however, the development of indurated swelling at the injection site limits vaccine acceptability to producers. This study determined whether a reduced dose of vaccine antigen, with a full dose of adjuvant, would produce comparable T cell-mediated immune responses with smaller lesions. T cell responses induced by in vitro stimulation with MAP antigen from calves vaccinated with full, half, and quarter doses of antigen were evaluated 2, 4, and 9 months after vaccination by multi-parameter flow cytometry (FCM) and the whole blood interferon-gamma (WB IFN-gamma) assay. The WB IFN-gamma responses were significantly elevated in vaccinated animals, but did not differ significantly between doses. FCM demonstrated antigen-specific responses for both IFN-gamma and IL-4 in the CD4 T cell population from vaccinated animals, while CD8 T cells and gammadelta T cells mainly responded with increased IFN-gamma. Dose may have affected some T cell subset parameters at some time points, but intradermal skin test responses, WB IFN-gamma production, IFN-gamma responses by T cell subsets in FCM were not significantly different between full, half, or quarter doses of antigen. Injection site lesions were smaller in animals vaccinated with a lower dose of antigen, but reached statistical significance (P<0.05) in the half dose group only.

Comparison of humoral and cellular immune responses to a pentavalent modified live virus vaccine in three age groups of calves with maternal antibodies, before and after BVDV type 2 challenge.

The aim of this study was to evaluate the ability of a pentavalent (BVDV types 1 and 2, BHV-1, BRSV, and PI-3) modified live virus (MLV) vaccine given to 1-2-, 4-5-, and 7-8-week-old calves with maternal antibodies to induce humoral and cellular immune responses and protect calves from virulent BVDV type 2. Eight calves in each age group were vaccinated and four served as controls. All calves were challenged intranasally with BVDV type 2, 12 weeks after vaccination. SVN titers to all five viruses declined in all groups after vaccination (except 4-5-week-old calves to BVDV type 1). After challenge, the SVN titers for both types of BVDV showed anamnestic responses in calves vaccinated at 4-5 and 7-8 weeks, but not at 1-2 weeks of age. In all groups, T cell subsets responded specifically to BVDV types 1 and 2 but not to BHV-1, BRSV, or PI-3 after vaccination by increasing their expression of activation markers (CD25, IFN-gamma and IL-4). All vaccinated calves were significantly protected from BVDV type 2 challenge.