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Abstract Background The cattle tick, Rhipicephalus (Boophilus) microplus, affects livestock production in many regions of the world. Up to now, the widespread use of chemical acaricides has led to the selection of acaricide-resistant ticks and to environmental contamination. Gavacplus is a subunit vaccine based on the recombinant Bm86 tick antigen expressed in yeast, capable to control infestations of R. microplus under controlled and production conditions. The vaccine constitutes the core element of broad control programs against this ectoparasite, in which acquired immunity in cattle to Bm86 is combined with a rational use of acaricides. At present, the conventional vaccine scheme consists of three doses that should be administered at weeks 0, 4 and 7, followed by a booster every six months. Results In this study we assayed a reduction in the number of the initial doses of Gavacplus, evaluated the time course and the level of bovine anti-Bm86 antibodies elicited, and analyzed the vaccine effect on ticks engorging on immunized cattle under production conditions. Following three different immunization schemes, the bovines developed a strong and specific immune response characterized by elevated anti-Bm86 IgG titers. A reduction in the weight of engorging female ticks, in the weight of the eggs laid and also in R. microplus viable eggs percentage was obtained by using only two doses of Gavacplus administered at weeks 0 and 4, followed by a booster six months later. This reduction did not differ from the results obtained on ticks engorging on cattle immunized at weeks 0, 4 and 7. It was also demonstrated that anti-Bm86 antibody titers over 1:640, measured in bovines immunized at weeks 0 and 4, were sufficient to affect weight and reproductive potential of female ticks as compared with ticks engorging on unvaccinated animals. In addition, no statistically significant differences were detected in the average weight of eggs laid by ticks engorged on immunized cattle that showed anti-Bm86 specific titers in the range of 1:640 to 1:81920. Conclusion The administration of two initial doses of Gavacplus containing 100 μg of Bm86 antigen to non-immunized cattle under production conditions is sufficient to affect the weight and the reproductive capacity of R. microplus engorging females. According to these results, cattle herds' manipulation and vaccine costs could be potentially reduced with a positive impact on the implementation of integrated control programs against R. microplus.

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.

Background: The differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear., Methods: Thymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation., Results: Despite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3 high CD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3 high CD8- thymocytes. As expected, CD3 high CD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1., Conclusion: FoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Swaminathan, Scorza, Yero, Farnos, Burke Schinkel, Angel and Jenabian.)

New influenza strains are constantly emerging, causing seasonal epidemics and raising concerns to the risk of a new global pandemic. Since vaccination is an effective method to prevent the spread of the disease and reduce its severity, the development of robust bioprocesses for producing pandemic influenza vaccines is exceptionally important. Herein, a membrane chromatography-based downstream processing platform with a demonstrated industrial application potential was established. Cell culture-derived influenza virus H1N1/A/PR/8/34 was harvested from benchtop bioreactor cultures. For the clarification of the cell culture broth, a depth filtration was selected as an alternative to centrifugation. After inactivation, an anion exchange chromatography membrane was used for viral capture and further processing. Additionally, two pandemic influenza virus strains, the H7N9 subtype of the A/Anhui/1/2013 and H3N2/A/Hong Kong/8/64, were successfully processed through similar downstream process steps establishing optimized process parameters. Overall, 41.3-62.5% viral recovery was achieved, with the removal of 86.3-96.5% host cell DNA and 95.5-99.7% of proteins. The proposed membrane chromatography purification is a scalable and generic method for the processing of different influenza strains and is a promising alternative to the current industrial purification of influenza vaccines based on ultracentrifugation methodologies.

CD8 T cells are key players in the clearance of human immunodeficiency virus (HIV)-infected cells, such that CD8 T-cell dysfunction contributes to viral persistence despite antiretroviral (ARV) therapy. Mesenteric lymph nodes (MLNs) are major sites of gut mucosal immunity. While different CD8 T cell subsets such as CD8 alpha-alpha (CD8αα), CD8 alpha-beta (CD8αβ), CD8 regulatory T cells (Treg), and mucosa-associated invariant T cells (MAIT) are present in the gut and exhibit distinct functions, their dynamics remain poorly understood due to the lack of accessibility to these tissues in humans. We thus assessed CD8 T cells in MLNs versus peripheral blood in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) following early ARV therapy initiation. SIV infection was associated with an increase over time of both CD8αβ and CD8αα T cells in the blood and MLNs, whereas early ARV initiation significantly decreased the frequencies of CD8αα but not CD8αβ T cells in MLNs. A significant decrease in the expression of chemokine receptors CCR6 and CXCR3 by CD8 T cells, which are essential for T-cell trafficking to the inflammatory sites, was observed in chronically SIV-infected RMs. Surprisingly, while MAIT cells are increased in ARV-treated RMs, their frequencies in MLN are extremely low and were not impacted by ARV. The acute infection resulted in an early CD39 + FoxP3 + CD8 Tregs increase in both compartments, which was normalized after early ARV. Frequencies of CD8 Treg cells were positively correlated with frequencies of CD4 Tregs and accordingly negatively correlated with the Th17/Treg ratio in the blood but not in MLNs. Overall, our results underscore the difference in CD8 T-cell subset dynamics in the blood and MLNs. IMPORTANCE Changes in CD8 T-cell subsets during acute SIV/HIV infections and following early ARV initiation in gut lymphoid tissues are poorly understood. Using an acute SIV infection model in rhesus macaques, we assessed the impact of early ARV, initiated 4 days postinfection, on relative proportions of CD8 T-cell subsets in MLNs compared to blood. We found that acute SIV infection and early ARV initiation differentially affect the distribution of effector CD8 T cells, CD8 MAIT cells, and CD8 Tregs in MLNs compared to blood. Overall, early ARV initiation maintains the frequency of effector CD8 T cells while reducing immunosuppressive CD39 + CD8 Tregs. Our study provides deeper insight into the dynamics of the CD8 T-cell compartment in gut mucosal immune surveillance during acute SIV infection and following early ARV initiation.

Background: HIV infection promotes the expansion of immunosuppressive regulatory T-cells (Tregs), contributing to immune dysfunction, tissue fibrosis and disease progression. Early antiretroviral treatment (ART) upon HIV infection improves CD4 count and decreases immune activation. However, Treg dynamics and their epigenetic regulation following early ART initiation remain understudied., Methods: Treg subsets were characterized by flow cytometry in 103 individuals, including untreated HIV-infected participants in acute and chronic phases, ART-treated in early infection, elite controllers (ECs), immunological controllers (ICs), and HIV-uninfected controls. The methylation status of six regulatory regions of the foxp3 gene was assessed using MiSeq technology., Findings: Total Treg frequency increased overtime during HIV infection, which was normalized in early ART recipients. Tregs in untreated individuals expressed higher levels of activation and immunosuppressive markers (CD39, and LAP(TGF-β1)), which remained unchanged following early ART. Expression of gut migration markers (CCR9, Integrin-β7) by Tregs was elevated during untreated HIV infection, while they declined with the duration of ART but not upon early ART initiation. Notably, gut-homing Tregs expressing LAP(TGF-β1) and CD39 remained higher despite early treatment. Additionally, the increase in LAP(TGF-β1) + Tregs overtime were consistent with higher demethylation of conserved non-coding sequence (CNS)-1 in the foxp3 gene. Remarkably, LAP(TGF-β1)-expressing Tregs in ECs were significantly higher than in uninfected subjects, while the markers of Treg activation and gut migration were not different., Interpretation: Early ART initiation was unable to control the levels of immunosuppressive Treg subsets and their gut migration potential, which could ultimately contribute to gut tissue fibrosis and HIV disease progression., Funding: This study was funded by the Canadian Institutes of Health Research (CIHR, grant MOP 142294) and in part by the AIDS and Infectious Diseases Network of the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S)., Competing Interests: Declaration of Competing Interest We have no competing of interest to declare., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Despite the success of antiretroviral therapy (ART) to control viral replication, people living with HIV (PWH) have high levels of chronic systemic inflammation and immune dysregulation which drives accelerated co-morbidities including coronary artery disease (CAD). Regulatory T cells (Tregs) and ectonucleotidases CD39/CD73 are known to be athero-protective via their immunosuppressive and anti-inflammatory functions., Design: We assessed the dynamics of Treg subsets in ART-treated PWH with or without CAD vs. HIV-uninfected individuals., Methods: Blood specimens were obtained from 142 participants including ART-treated HIV-infected adults with (n = 43) or without CAD (n = 41), as well as HIV-uninfected controls with (n = 31) or without CAD (n = 27). CAD was determined by the presence of atherosclerotic features on computed tomography angiography of the coronary arteries performed on all study participants. Treg subsets frequencies were assessed by flow cytometry., Results: Regardless of statin treatment or ART regimen, HIV+CAD+ individuals had the highest total Treg frequencies and increased thymic generation and output of Tregs (Helios/CD31 expression), while athero-protective CD39+/CD73+ Tregs were significantly depleted in this group. Tregs from PWH had higher expression of CCR6/CXCR3 than uninfected individuals regardless of CAD, while in HIV+CAD+ individuals Tregs expressed the highest levels of CCR4, which limits their maintenance. The lowest levels of CD4+ and CD8+ T-cell immune activation has been observed in HIV+CAD+ within study groups., Conclusion: ART-treated PWH with diagnosed CAD are characterized by profound alterations in populations of anti-inflammatory and athero-protective Treg subsets. These changes may contribute to atherosclerotic plaque formation and progression during chronic HIV infection in the ART era., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Background: Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) is involved in immune dysregulation during HIV infection. Niacin (vitamin B3) could control the excess of tryptophan depletion and represents a potential strategy to improve immune functions and CD4 count recovery in immunological non-responder HIV-infected individuals on antiretroviral therapy (ART)., Methods: Methods: In the CTN PT006 phase 2 pilot randomized trial, 20 adults on ART with CD4 ≤ 350 cells/µl, despite an undetectable viral load (VL) for at least 3 months, received 2000 mg of extended-release (ER)-niacin orally once daily for 24 weeks. Side effects, VL, CD4/CD8 counts, lipid profile, T-cell activation and senescence, Tregs and Th17 cell frequencies, Kyn/Trp ratio, and levels of IL-6, IP-10, sST2, I-FABP, and LBP were assessed following ER-niacin treatment., Results: Results: Thirteen participants completed the study. Treatment was interrupted in 4 patients due to loss of follow-up or personal reasons and 3 patients were discontinued due to comorbidity risks. All participants maintained a VL < 40 copies/ml, while ER-niacin did not affect CD4 and CD8 cell counts. Plasma levels of triglycerides, total, and LDL cholesterol significantly decreased, following ER-niacin treatment. ER-niacin also diminished Kyn plasma levels and slightly decreased CD4 T-cell activation. However, no improvement in CD8 subsets, Kyn/Trp ratio, Th17/Treg balance, and plasma inflammatory markers was observed., Conclusions: Conclusions: Although ER-niacin combined with ART was well-tolerated among immune non-responders and decreased plasma lipids, it did not improve systemic inflammation, Kyn/Trp ratio, and CD4 cell recovery. Overall, ER-niacin was not effective to overcome chronic inflammation in PLWH.

The lungs are relatively unexplored anatomical human immunodeficiency virus (HIV) reservoirs in the antiretroviral therapy (ART) era. Double negative (DN) T cells are a subset of T cells that lack expression of CD4 and CD8 (CD4 - CD8 - ) and may have both regulatory and effector functions during HIV infection. Notably, circulating DN T cells were previously described as cellular HIV reservoirs. Here, we undertook a thorough analysis of pulmonary versus blood DN T cells of people living with HIV (PLWH) under ART. Bronchoalveolar lavage (BAL) fluid and matched peripheral blood were collected from 35 PLWH on ART and 16 uninfected volunteers without respiratory symptoms. Both PLWH and HIV-negative (HIV - ) adults displayed higher frequencies of DN T cells in BAL versus blood, and these cells mostly exhibited an effector memory phenotype. In PLWH, pulmonary mucosal DN T cells expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3, and PD-1) than blood. We also observed that DN T cells were less senescent (CD28 - CD57 + ) and expressed less immunosuppressive ectonucleotidase (CD73/CD39), granzyme B, and perforin in the BAL fluid than in the blood of PLWH. Importantly, fluorescence-activated cell sorter (FACS)-sorted DN T cells from the BAL fluid of PLWH under suppressive ART harbored HIV DNA. Using the humanized bone marrow-liver-thymus (hu-BLT) mouse model of HIV infection, we observed higher infection frequencies of lung DN T cells than those of the blood and spleen in both early and late HIV infection. Overall, our findings show that HIV is seeded in pulmonary mucosal DN T cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART. IMPORTANCE Reservoirs of HIV during ART are the primary reasons why HIV/AIDS remains an incurable disease. Indeed, HIV remains latent and unreachable by antiretrovirals in cellular and anatomical sanctuaries, preventing its eradication. The lungs have received very little attention compared to other anatomical reservoirs despite being immunological effector sites exhibiting characteristics ideal for HIV persistence. Furthermore, PLWH suffer from a high burden of pulmonary non-opportunistic infections, suggesting impaired pulmonary immunity despite ART. Meanwhile, various immune cell populations have been proposed to be cellular reservoirs in blood, including CD4 - CD8 - DN T cells, a subset that may originate from CD4 downregulation by HIV proteins. The present study aims to describe DN T cells in human and humanized mice lungs in relation to intrapulmonary HIV burden. The characterization of DN T cells as cellular HIV reservoirs and the lungs as an anatomical HIV reservoir will contribute to the development of targeted HIV eradication strategies., (Copyright © 2020 Meziane et al.)

Increased frequencies of immunosuppressive regulatory T cells (Tregs) are associated with gut lymphoid tissue fibrosis and dysfunction which, in turn, contribute to disease progression in chronic simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection. Mesenteric lymph nodes (MLNs), which drain the large and small intestine, are critical sites for the induction and maintenance of gut mucosal immunity. However, the dynamics of Tregs in MLNs are not well understood due to the lack of accessibility to these tissues in HIV-infected individuals. Here, the dynamics of Tregs in blood and MLNs were assessed in SIV-infected rhesus macaques (RMs) following early antiretroviral drug (ARV) initiation. Early ARV initiation reduced T-cell immune activation, as assessed by HLA-DR/CD39 expression, and prevented the depletion of memory CCR6 + Th17 cells in both blood and MLNs. Untreated animals showed higher frequencies of Tregs, CD39 + Tregs, thymic Tregs, and new memory CD4 populations sharing similarity with Tregs as CTLA4 + PD1 - and CTLA4 + PD1 - FoxP3 + T cells. Despite early ARV treatment, the frequencies of these Treg subsets remained unchanged within the MLNs and, in contrast to blood normalization, the Th17/Treg ratio remained distorted in MLNs. Furthermore, our results highlighted that the expressions of IDO-1, TGFβ1 and collagen-1 mRNA remained unchanged in MLN of ARV-treated RMs. ARV interruption did not affect T-cell immune activation and Th17/Treg ratios in MLN. Altogether, our data demonstrated that early ARV initiation within the first few days of SIV infection is unable to reduce the frequencies and homing of various subsets of Tregs within the MLNs which, in turn, may result in tissue fibrosis, impairment in MLN function, and HIV persistence. IMPORTANCE Tregs contribute to SIV/HIV disease progression by inhibition of antiviral specific responses and effector T-cell proliferation. Tregs also cause tissue fibrosis via transforming growth factor β1 production and collagen deposition, which are associated with microbial translocation and generalized immune activation. Early ARV initiation upon viral exposure is recommended globally and results in improved immune function recovery and reduced viral persistence. Here, using an acute SIV infection model of rhesus macaques, we demonstrated for the first time that despite clear improvements in mucosal CD4 T cells, in contrast to blood, Treg frequencies in MLNs remained elevated following early ARV initiation. The particular Th17/Treg balance observed in MLNs can contribute, in part, to the maintenance of mucosal fibrosis during suppressive ARV treatment. Our results provide a better understanding of gut mucosal immune dynamics following early ARV initiation. These findings suggest that Treg-based treatments could serve as a novel immunotherapeutic approach to decrease gut mucosal damage during SIV/HIV infections., (Copyright © 2019 Yero et al.)

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Background: The lungs were historically identified as one of the major anatomic sites for HIV replication in the pre-antiretroviral therapy (ART) era. However, their contribution to HIV persistence in individuals under suppressive ART remains understudied., Design: We assessed HIV persistence and comprehensively characterized pulmonary mucosal CD4 T cells in HIV-infected (HIV) individuals receiving long-term suppressive ART versus uninfected participants., Methods: Bronchoalveolar lavage (BAL), bronchial biopsies, and matched peripheral blood were obtained from n = 24 HIV-infected adults receiving long-term suppressive ART (median: 9 years) and n = 8 healthy volunteers without respiratory symptoms. HIV-DNA and cell-associated HIV-RNA were quantified by ultra-sensitive PCR, and lung mucosal CD4 T-cell subsets were characterized by multiparameter flow cytometry., Results: The levels of HIV-DNA were 13-fold higher in total BAL cells compared to blood. Importantly, FACS-sorted CD4 T cells from BAL contained greater levels of HIV-DNA compared to peripheral CD4 T cells. BAL CD4 T cells in HIV individuals were characterized mostly by an effector memory phenotype, whereas naive and terminally differentiated cells were underrepresented compared to blood. Furthermore, BAL CD4 T cells expressed higher levels of immune activation (HLA-DR/CD38) and senescence (CD57) markers. Importantly, BAL was enriched in T-cell subsets proposed to be preferential cellular HIV reservoirs, including memory CD4CCR6, Th1Th17 (CD4CCR6CCR4CXCR3), CD4CCR6CXCR3CCR4, and CD4CD32a T cells., Conclusion: The pulmonary mucosa represents an important immunological effector site highly enriched in activated and preferential CD4 T-cell subsets for HIV persistence during long-term ART in individuals without respiratory symptoms. Our findings raise new challenges for the design of novel HIV eradication strategies in mucosal tissues.

Numerous studies in chordates and arthropods currently indicate that Cdx proteins have a major ancestral role in the organization of post-head tissues. In urochordate embryos, Cdx loss-of-function has been shown to impair axial elongation, neural tube (NT) closure and pigment cell development. Intriguingly, in contrast to axial elongation and NT closure, a Cdx role in neural crest (NC)-derived melanocyte/pigment cell development has not been reported in any other chordate species. To address this, we generated a new conditional pan-Cdx functional knockdown mouse model that circumvents Cdx functional redundancy as well as the early embryonic lethality of Cdx mutants. Through directed inhibition in the neuroectoderm, we provide in vivo evidence that murine Cdx proteins impact melanocyte and enteric nervous system development by, at least in part, directly controlling the expression of the key early regulators of NC ontogenesis Pax3,Msx1 and Foxd3 Our work thus reveals a novel role for Cdx proteins at the top of the trunk NC gene regulatory network in the mouse, which appears to have been inherited from their ancestral ortholog., (© 2016. Published by The Company of Biologists Ltd.)

E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3' terminus. The recombinant antigen was produced at a range of 120-150 microg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 microg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 10(5) PLD(50) of homologous CSFV "Margarita" strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.

Classical swine fever virus is the etiological agent of the most economically important highly contagious disease of swine worldwide. E2 is the major envelope glycoprotein present as a homodimer on the outer surface of the virus and represents an important target for the induction of neutralizing immune response against the viral infection. The E2 extracellular domain was expressed in the milk of adenoviral transduced goats at the highest level about 1.2g/L. The recombinant glycoprotein was purified from clarified serum milk by a single metal chelate affinity chromatography step, as a homodimer of approximately 100kDa and purity over 98%. Glycosylation analysis showed the presence of oligomannoside, hybrid and complex type N-glycans, attached to the recombinant E2. The capacity of goat milk-derived E2 antigen to protect pigs from both classical swine fever clinical signs and viral infection was assessed in a vaccination and challenge trial. The immunized pigs became protected after challenge with 10(5) LD(50) of a highly pathogenic CSFV strain. In the context of veterinary vaccines, this expression system has the advantages that the recombinant antigen could be harvested in about 48h after adenoviral transduction with expression levels in the range of g/L. This approach may turn into a scalable expression system for the assessment and production of veterinary vaccines.