Type I IFN modulates the immune response induced by DNA vaccination to pseudorabies virus glycoprotein C.

DNA vaccines have the capacity to induce strong Th1-biased immune responses that are of major importance to providing protection against intracellular pathogens. In the present study we have focused on the role played by type I IFN in immune responses induced after DNA vaccination. Mice lacking the IFNAR1 chain of the type I IFN receptor (IFNAR K/O mice) were immunized with a plasmid encoding glycoprotein C of pseudorabies virus (PRV-gC). After DNA vaccination, wild-type (WT) mice showed features characteristic of Th1 immune responses, such as high IgG2a:IgG1 anti-PRV Ab ratio and antigen-specific IFN-gamma production by spleen cells. In contrast, IFNAR K/O mice showed a significantly lower IgG2a:IgG1 Ab ratio and IFN-gamma production. In addition, the percentage of CD8(+) and B lymph-node cells expressing CD69 after PRV-gC DNA vaccination was lower in IFNAR K/O than in WT mice. These results support a major role played by type I IFN in shaping Th1 immune responses after DNA vaccination. Codelivery of plasmids encoding IL-12 and IL-18 along with the plasmid encoding PRV-gC restored Th1 responses in IFNAR K/O mice.

Interferon-alpha-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets.

Transmissible gastroenteritis virus (TGEV) infection of piglets results in a very rapid and massive release of IFN-alpha in serum and secretions. The objective of this work was to characterize the IFN-alpha-producing cells (IPC) in tissues of TGEV-infected piglets. Caesarean-derived colostrum-deprived piglets were infected orally with the TGEV virulent Miller strain and IPC were characterized in situ by immunohistochemistry, using a rabbit anti-pig IFN-alpha antiserum. IPC were almost exclusively detected in intestinal tissues and mesenteric lymph nodes (MLN), as early as 6 h post inoculation (p.i.), with a peak at 12-18 h. They disappeared by 24 h. IPC were localized between enterocytes in the small intestine epithelial layer, in the lamina propria, around the Peyer's patches and, at highest frequency, in MLN. Very few IPC were present in the spleen and popliteal lymph nodes of infected piglets. Double immunohistochemical staining for IFN-alpha and leukocyte markers on MLN cryosections showed that IPC were mainly Swine Leukocyte Antigen (SLA) class II positive, and were not stained by an anti-macrophage (SWC3a) MAb. In addition, double staining with anti-TGEV and anti-IFN-alpha MAbs showed that viral antigens were present in MLN, close to IPC. These results show for the first time the presence of IPC in gut mucosa and gut-associated lymphoid tissues in response to an enteropathogenic virus. Moreover, this work shows that IFN-alpha released in serum is likely to originate almost exclusively from gut IPC triggered locally by viral antigens to produce IFN-alpha, since there were very few IPC in spleen or peripheral lymph nodes. MHC class II molecule expression by gut-associated IPC suggests that these cells may be the in vivo mucosal counterparts of the dendritic cells recently shown to produce IFN-alpha after in vitro viral induction.

Transient IFN-gamma synthesis in the lymph node draining a dermal site loaded with UV-irradiated herpes simplex virus type 1: an NK- and CD3-dependent process regulated by IL-12 but not by IFN-alpha/beta.

Our previous studies have shown that UV-inactivated, non-replicating herpes simplex virus type 1 (UV-HSV-1) triggers early and transient synthesis of IFN-alpha/beta in the mouse regional lymph node when delivered upstream (i.e. in the ear dermis). In this study, it is demonstrated, by use of a quantitative RT-PCR readout assay, that IFN-gamma mRNA expression was rapidly and transiently upregulated in draining lymph nodes when UV-HSV-1 was delivered in the ear dermis of C57Bl/6 mice. An increased number of IFN-gamma-producing cells was also detected in the lymph node by flow cytometric analysis. Two different subsets of cells, namely DX5(+) NK cells and CD3epsilon(+) T cells, accounted for this early IFN-gamma synthesis. Prompt upregulation of IFN-alpha and IL-12p40 mRNA was also recorded. We took advantage of IFN-alpha/beta-receptor knockout and wild-type 129 mice to study a potential role of IFN-alpha/beta in the signalling pathway leading to IFN-gamma transcription/translation. IFN-gamma mRNA upregulation still occurred in IFN-alpha/beta-receptor(-/-) mice, showing that IFN-alpha/beta was dispensable. The use of IL-12-neutralizing antibodies, prior to UV-HSV-1 delivery, confirmed the major role played by IL-12 in the early/transient IFN-gamma burst.

Interferon alpha inducing property of coronavirus particles and pseudoparticles.

Previous work in our laboratory have provided evidence that the membrane glycoprotein M of TGEV is centrally involved in efficient induction of alpha interferon (IFN-alpha) synthesis by non-immune peripheral blood mononuclear cells incubated with fixed, TGEV-infected cells or inactivated virions. Here we report recent completion of studies initiated to get a better understanding of the nature of the interferogenic determinant(s). Transfected cells expressing TGEV M together with the minor structural component E (formerly called sM) were found to trigger IFN-alpha synthesis. Co-expression of these two proteins was shown to be necessary and sufficient for assembly and release of pseudoparticles resembling TGEV virions. Purified pseudoparticles exhibited an interferogenic activity close to that of authentic virions. Chimeric recombinant particles expressing BCV M ectodomain also induced IFN. Examination of cell cultures infected by viruses representative of the three Nidovirales genera revealed that the capacity to act as an efficient IFN-alpha inducer is a common feature of viral particles of the coronavirus genus. Altogether these data bring new insights regarding the putative nature of the viral structure involved in IFN-alpha induction.

Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes.

Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-alpha synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-alpha-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-alpha almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-alpha is more likely to involve a specific, multimeric structure than a definite sequence motif.

Interferon-alpha response to swine arterivirus (PoAV), the porcine reproductive and respiratory syndrome virus.

We studied the interferon-alpha (IFN-alpha) system in relation to the porcine arterivirus (PoAV), porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant porcine IFN-alpha inhibited the growth of this virus in alveolar macrophage cultures. When pigs were challenged intranasally with PoAV, their serum contained IFN-alpha in relatively low concentrations on the second day after challenge and up to 5 days at the latest. Most animals had no IFN-alpha in their lung secretions, even though PoAV replicates in the respiratory tract. In vitro, PoAV replicates in alveolar macrophages, but neither these nor peripheral blood mononuclear cells (PBMC) produced IFN-alpha in response to infection. This may be because PoAV suppresses IFN-alpha production. When macrophages treated with PoAV were superinfected with swine transmissible gastroenteritis virus (TGEV), a known good inducer of IFN, no IFN-alpha was detected. This suppressive effect was lost when the virus was inactivated by UV light. Our results suggest that downregulation of IFN-alpha production may play an important part in enabling PoAV to replicate in cell cultures and in pigs.

In vivo induction of interferon-alpha in pig by non-infectious coronavirus: tissue localization and in situ phenotypic characterization of interferon-alpha-producing cells.

A low frequency peripheral blood mononuclear cell (PBMC) subpopulation, referred to as natural interferon-producing (NIP) cells, is described as producing interferon-alpha (IFN-alpha) following contact with non-infectious viral structures, namely viral glycoproteins. These cells are characterized in vitro as non-T, non-B, MHC class II+ and CD4+ cells. In this study, NIP cells were analysed in vivo after an intravenous injection of UV-inactivated transmissible gastroenteritis virus in newborn piglets, which resulted in strong serum IFN-alpha production. Splenocytes, but not PBMC, were the IFN-alpha producers in vivo. Using double immunohistochemical labelling for both IFN-alpha and leukocyte markers, we established that splenic NIP cells were not T or B cells. The majority were MHC class II+ and only a minority expressed a macrophage marker. NIP cells were localized in contact with MHC class II-expressing cells and T cells, which suggested that NIP cells might modulate the antiviral immune response.

Herpes simplex virus induces appearance of interferon-alpha/beta-producing cells and partially interferon-alpha/beta-dependent accumulation of leukocytes in murine regional lymph nodes.

As in vivo experimental system involving the local induction of interferon-alpha/beta (IFN-alpha/beta) responses was established in mice by injecting s.c. ultraviolet (UV)-inactivated herpes simplex virus (HSV) in the right ears, the left ears receiving phosphate-buffered saline (PBS) as a control. Circulating IFN-alpha/beta was present in blood as early as 6 h postinjection, and little or none was found 24 h postinjection. Identification of IFN-alpha/beta-producing cells, carried out by immunohistochemistry and in situ hybridization, demonstrated that the IFN response occurred mainly in the lymph node draining the HSV-injected ear and not in the contralateral lymph node. Occasionally, IFN-alpha/beta-producing cells were found in the spleen and in the skin. The injected HSV caused an inflammatory reaction in the skin and an almost threefold enlargement of the draining lymph node within 6 h. The latter was characterized by a general accumulation of all major lymphocyte subsets and a striking infiltration of neutrophils. Injection s.c. of neutralizing anti-IFN-alpha/beta antibodies before HSV injection reduced the increase in size of the draining lymph node by approximately 50% at 6 h, and no significant effects were seen at 24 h. The localization of cells producing IFN-alpha/beta in the lymph node and the capacity of such IFN-alpha/beta to at least partially mediate an early accumulation of cells suggest that the local IFN-alpha/beta response may have an important role in the initiation of early antiviral immune responses.

Identification of Schistosoma haematobium, S. bovis and S. curassoni by multivariate analysis of cercarial papillae indices.

The disposition of cercarial papillae of 68 pre-identified Schistosoma species was established. All the cercariae originated from Africa and Madagascar and were either obtained from natural or experimental infections, and belonged to three species Schistosoma haematobium, S. bovis and S. curassoni. Discriminant analysis was based on nine characters: average values, skewness and kurtosis of three cercarial indices (AD, AL and U) for each sample or isolate. AD, AL correspond respectively to the relative distance between dorsal and lateral papillae. U corresponds to the total number of tail stem papillae. With the exception of two cases of the 68 (one of them corresponding to cercariae shed by a non-African experimentally infected snail), the method enabled discrimination of S. haematobium, S. bovis and S. curassoni.

[Development of the filaria Monanema martini in the epidermis of ixodid ticks]. / Développement de la filaire Monanema martini dans l'épiderme des tiques ixodidae.

Morphological and histological analysis of the larval development of Monanema martini, a filaria with skin dwelling microfilariae. The vectors are the hexapod larvae of Rhipicephalus sanguineus, R. turanicus and H. truncatum; the filarial infective stages appear during the larval-nymph moult of the vector (11 days at 26 degrees C). This species, and in our opinion the other species of Monanema, have a complete development in the epidermis of the ticks. Hard-ticks (Ixodidae) appear to be the main vectors of filariae with skin-dwelling microfilariae belonging to Dipetalonema evolutionary line: Yatesia, Cherylia, Cercopithifiliaria, Monanema.

[Interference between anthelmintics and immunity in gastrointestinal strongylosis of sheep]. / Interférence entre vermifugation et immunité dans les strongyloses gastro-intestinales du mouton.

Groups of very similar sheep (breed, sex, age, hemoglobin type, family) were immunised against H. contortus by repeated infections. Self-cure and immune reactions expelled parasites but some of them still remain in the gut of animals; they have been eliminated by dosage of anthelmintics. Animal were challenged; a discrepancy exists between numbers of eggs passed in each group (immune and dosed animals, immune and non-dosed animals and non-immune animals); the best fitting estimated equation on experimental values (or on moving average points) is used for statistical tests. Immunity vanishes partly (or totally) when animals have been dosed; a residual population of worms is thought to be responsible for immunity because of a permanent booster.

[Biological assay of anthelmintic drugs. Determination of DE 50]. / Essais biologiques des médicaments anthelminthiques. Détermination de la DE 50.

Experiments on ivermectine, thiabendazole and levamisole were performed by using the Nippostrongylus brasiliensis rat model. The aim of the experiments was to study the 50% as well as 99.5% doses of the above anthelmintics. Besides this a relative evaluation of drugs was also carried out and different dose efficiencies were compared by keeping one anthelmintic as a standard. The results were obtained on the following experimental conditions: homogeneity of the group of rats (age, weight and sex), uniform infestation in all the animals (3000 larvae at day zero), administration of anthelmintics was done on day 6 and autopsy was performed on the 9th day of infection. The effectivity of anthelmintic is essentially based on the mortality of the adult worms. The mathematical expression is obtained on the basis of logarithmic values of the doses and probit percentage analysis. The graph obtained between dose vs efficiency is a straight line, so the value of DE 50 can be calculated using this straight line. The normal distribution of worms exposed to a dose was verified which justifies the statistical interpretation.