Safety study of Rift Valley Fever human vaccine candidate (DDVax) in mosquitoes.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen with significant human and veterinary health consequences that periodically emerges in epizootics. RVFV causes fetal loss and death in ruminants and in humans can lead to liver and renal disease, delayed-onset encephalitis, retinitis, and in some cases severe haemorrhagic fever. A live attenuated vaccine candidate (DDVax), was developed by the deletion of the virulence factors NSs and NSm from a clinical isolate, ZH501, and has proven safe and immunogenic in rodents, pregnant sheep and non-human primates. Deletion of NSm also severely restricted mosquito midgut infection and inhibited vector-borne transmission. To demonstrate environmental safety, this study investigated the replication, dissemination and transmission efficiency of DDVax in mosquitoes following oral exposure compared to RVFV strains MP-12 and ZH501. Infection and dissemination profiles were also measured in mosquitoes 7 days after they fed on goats inoculated with DDvax or MP-12. We hypothesized that DDVax would infect mosquitoes at significantly lower rates than other RVFV strains and, due to lack of NSm, be transmission incompetent. Exposure of Ae. aegypti and Cx. tarsalis to 8 log10 plaque forming units (PFU)/ml DDVax by artificial bloodmeal resulted in significantly reduced DDVax infection rates in mosquito bodies compared to controls. Plaque assays indicated negligible transmission of infectious DDVax in Cx. tarsalis saliva (1/140 sampled) and none in Ae. aegypti saliva (0/120). Serum from goats inoculated with DDVax or MP-12 did not harbour detectable infectious virus by plaque assay at 1, 2 or 3 days post-inoculation. Infectious virus was, however, recovered from Aedes and Culex bodies that fed on goats vaccinated with MP-12 (13.8% and 4.6%, respectively), but strikingly, DDvax-positive mosquito bodies were greatly reduced (4%, and 0%, respectively). Furthermore, DDVax did not disseminate to legs/wings in any of the goat-fed mosquitoes. Collectively, these results are consistent with a beneficial environmental safety profile.

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis.

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (M Phi) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDM Phi) as target cells. Various B. abortus antigen preparations were tested including whole gamma-irradiated B. abortus bacteria (gamma BA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDM Phi targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with gamma BA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4+, CD8(-) cells indicating that the observed cytotoxic responses were most likely due to an inflammatory Th1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.

Recombinant bovine interleukin 2 enhances immunity and protection induced by Brucella abortus vaccines in cattle.

Augmentation of immunization of cattle Brucella abortus S19 or a B. abortus soluble protein extract (SPEBA) vaccine through administration of recombinant bovine IL 2 (rBoIL 2) was evaluated. Seventy-five heifers were divided among 6 groups that were treated with the following: Group 1, no treatment; Group 2, rBoIL 2 (1microg/kg) on day 0; Group 3, SPEBA (2 mg) on day 0 and week 9; Group 4, SPEBA + rBoIL 2 on day 0, SPEBA on week 9; Group 5, S19 (10(7) CFU) on day 0 and week 9; Group 6, S19 + rBoIL 2 on day 0, S19 only on week 9. Approximately, 6 months after vaccination, cattle were bred by natural service, and at mid-gestation pregnant cattle were challenged intraconjunctivally with 9.1 x 10(5) CFU of virulent B. abortus S2308. Pre- and post-challenge antibody responses were measured by an enzyme-linked immunosorbent assay, a particle concentration fluorescence assay, and the card test. Lymphoproliferation (LP) responses to gamma-irradiated B. abortus and SPEBA antigens were measured in peripheral blood mononuclear cells. After vaccination, antibody responses to B. abortus elevated rapidly in SPEBA- and S19-vaccinates with and without rBoIL 2, however, these responses were significantly (P < 0.05) higher in vaccinates which also received rBoIL 2. Antibody levels for all vaccinated groups had returned to those of negative control groups by the challenge date with the exception of the SPEBA/rBoIL 2 group. In general, LP responses were higher in vaccinated or rBoIL 2-treated cattle than for unvaccinated controls. Challenge of 48 pregnant heifers resulted in abortions in 4/9 of Group 1, 0/9 of Group 2, 4/8 of Group 3, 2/9 of Group 4, 1/7 of Group 5, and 0/6 of Group 6 cattle. Treatment with rBoIL 2 alone (Group 2) provided significant (P < 0.05) protection from infection, abortions and induction of sero-positive status compared to untreated (Group 1) cattle. Co-administration of rBoIL 2 with S19 resulted in significant (P < 0.05) augmentation in onset, duration and magnitude of LP responses to B. abortus antigens following challenge. Characterization of the cytokine response of bovine monocyte-derived macrophages by real-time polymerase chain reaction indicated that in vitro stimulation of these cells with rBoIL 2 resulted in a profound up-regulation of genes encoding tumor necrosis factor-alpha, IL 12p40, and interferon-gamma reflecting activation of the cells. Overall, rBoIL 2-treatment was associated with fewer infections, sero-conversions and a significant (P = 0.02) level of protection against abortion as compared to vaccination alone or no treatment.

Bovine T lymphocyte responses to Brucella abortus.

The long-held paradigm of T lymphocyte-mediated activation of mononuclear phagocytes (Mø) as the major mechanism of protection against facultative intracellular pathogens such as Brucella has been modified to include killing of infected Mø by various subsets of T lymphocytes. Remnants of killed infected cells are phagocytosed by immunologically-activated Mø, which are much more efficient at killing such pathogens. Most of the detailed information regarding immunity in general and that of brucellosis specifically has been obtained using murine infection models rather than in cattle. However, there has been considerable definition of cellular phenotypes, cytokines and functional characteristics of T lymphocytes in cattle over the last decade. This was mainly due to development of monoclonal antibodies against cell surface markers and application of molecular cloning and polymerase chain reaction (PCR) for isolation, characterization and detection of genes encoding bovine cytokines. This review discusses cellular and molecular immunity in bovine brucellosis as pertains to T lymphocyte interactions with the Mø. Although current knowledge directly obtained from brucellosis immunity studies in the bovine host is limited and incomplete, the many parallels between the bovine and murine immune systems allow for some extrapolation in the description of bovine host defense mechanisms. Direct information from studies with immunized cattle supports the concepts of coordinate activation of uninfected Mø and killing of Brucella-infected Mø by antigen-specific T lymphocytes as major mechanisms of host defense in bovine brucellosis. There also appears to be a bias in the T lymphocyte compartment towards recognition of particular bacterial stress proteins following immunization with live Brucella vaccines.

Cytokine expression by pulmonary leukocytes from calves challenged with wild-type and leukotoxin-deficient Mannheimia haemolytica.

The objective of this study was to assess the role of leukotoxin (LKT) in modulating the pulmonary cytokine response of calves challenged with Mannheimia haemolytica. Thirty-six calves, seronegative to LKT and M. haemolytica whole cell antigen were divided into three groups (I, II and III). Calves in groups I and II were challenged by the intra-bronchial route with 25 mL of phosphate buffered saline (PBS) containing 0.44×10(9) cfu/mL of LKT deficient (lkt(-)) and 25 mL of PBS containing 0.31×10(9) cfu/mL of wild-type (wt) M. haemolytica serotype 1, respectively. Group III calves were challenged intra-bronchially with 25 mL of sterile PBS. Leukocytes were collected from broncho-alveolar lavage fluid (BALF) 4 days before and at 1, 3, and 6 days post-inoculation (p.i.). Expression of the following cytokines in the recovered leukocytes was measured using real-time PCR: interleukin (IL)-1ß, -8, -10, -12 (p40) and TNF-α. The amount of TNF-α produced was also quantified by ELISA. Although a statistically significant difference in the expression of these cytokines was not observed between groups challenged with the wt and lkt(-) strains, the wt infected group (II) did exhibit higher mean clinical scores. Overall, there was considerable variation in the composition of the BALF between the groups and by day 7 p.i., both lkt(-)- and wt-challenged calves had seroconverted to M. haemolytica.

Cytotoxicity and cytokine production by bovine alveolar macrophages challenged with wild type and leukotoxin-deficient Mannheimia haemolytica.

Leukotoxin (LKT) is a virulence factor for Mannheimia haemolytica. In this study, bovine alveolar macrophages (BAMs) were challenged with wild type (wt) and LKT deficient (lkt(-)) M. haemolytica at a concentration of 1 bacterium/BAM and the cytokine response was quantified by ELISA and real-time reverse transcriptase-PCR. Significant increases in protein concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-10 were observed in supernatants obtained from BAMs challenged with the lkt(-) strain of M. haemolytica compared with wt challenged BAMs. There were no significant differences in mRNA expression of TNFα, IL-1ß, IL-6, IL-8 or IL-10 between BAMs challenged with the lkt(-) strain of M. haemolytica compared with wt challenged BAMs. BAMs challenged with the wt strain exhibited, on average, 43% more cytotoxicity than lkt(-) challenged BAMs (P<0.01).

Vaccination with Pasteurella multocida recombinant OmpA induces strong but non-protective and deleterious Th2-type immune response in mice.

Pasteurella multocida OmpA (PmOmpA) belongs to the major and multifunctional Escherichia coli OmpA family of proteins. We have previously reported that the protein is conserved, immunogenic and an adhesin that binds host cells and host cell extracellular matrix molecules [Dabo SM, Confer AW, Quijano-Blas RA. Molecular and immunological characterization of Pasteurella multocida serotype A:3 OmpA: evidence of its role in P. multocida interaction with extracellular matrix molecules. Microb Pathog 2003;35(4):147-157]. In this study, we found that immunization of mice with the recombinant PmOmpA elicited strong Th2-type immune response, characterized by high immunoglobulin G(1) (IgG(1)) antibodies production. Subsequent intraperitoneal homologous challenge of the immunized mice resulted in lack of protection associated with the high IgG(1) titers in anti-rPmOmpA sera. Furthermore, the protection afforded by vaccination with P. multocida OMPs alone was adversely affected by the addition of the rPmOmpA to the vaccine preparations. The results demonstrate that PmOmpA has a detrimental effect on the efficacy of vaccination with OMPs in mice. Targeted inactivation of pmOmpA gene in P. multocida 232 represents a potential mean towards the development of an effective vaccine candidate.