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Highly pathogenic avian influenza viruses (HPAIVs) cause severe systemic disease and high mortality rates in chickens, leading to a huge economic impact in the poultry sector. However, some chickens are resistant to the disease. This study aimed at evaluating the mechanisms behind HPAIV disease resistance. Chickens of different breeds were challenged with H7N1 HPAIV or clade 2.3.4.4b H5N8 HPAIV, euthanized at 3 days post-inoculation (dpi), and classified as resistant or susceptible depending on the following criteria: chickens that presented i) clinical signs, ii) histopathological lesions, and iii) presence of HPAIV antigen in tissues were classified as susceptible, while chickens lacking all these criteria were classified as resistant. Once classified, we performed RNA-Seq from lung and spleen samples in order to compare the transcriptomic signatures between resistant and susceptible chickens. We identified minor transcriptomic changes in resistant chickens in contrast with huge alterations observed in susceptible chickens. Interestingly, six differentially expressed genes were downregulated in resistant birds and upregulated in susceptible birds. Some of these genes belong to the NF-kappa B and/or mitogen-activated protein kinase signaling pathways. Among these six genes, the serine protease-encoding gene PLAU was of particular interest, being the most significantly downregulated gene in resistant chickens. Expression levels of this protease were further validated by RT-qPCR in a larger number of experimentally infected chickens. Furthermore, HPAIV quasi-species populations were constructed using 3 dpi oral swabs. No substantial changes were found in the viral segments that interact with the innate immune response and with the host cell receptors, reinforcing the role of the immune system of the host in the clinical outcome. Altogether, our results suggest that an early inactivation of important host genes could prevent an exaggerated immune response and/or viral

African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71DCD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding b-glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71DCD2 as a template: The single mutant BA71DCD2f, this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71DCD2f, BA71DCD2DP96R and BA71DCD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71DCD2f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future.

[Importance] Therapies targeting the programmed cell death 1 (PD-1) receptor or its ligand (PD-L1), such as the humanized monoclonal antibody durvalumab, have shown durable clinical responses in several tumor types. However, concerns about the safety and feasibility of PD-1/PD-L1 blockade in HIV-1–infected individuals have led to the exclusion of these patients from clinical trials on cancer immunotherapies., [Objective] To evaluate the feasibility and safety of durvalumab treatment in patients with advanced cancer and virologically controlled HIV-1 infection., [Design, Setting, and Participants] The DURVAST study was a nonrandomized, open-label, phase 2 clinical trial in patients with any solid tumor type in which anti–PD-1 or anti–PD-L1 antibodies have approved indications or for which there are data of antitumoral activity with no other available curative therapy. All patients had basal undetectable plasma viremia while undergoing combination antiretroviral therapy., [Interventions] Treatment consisted of intravenous infusion of durvalumab (1500 mg every 4 weeks) until disease progression or unacceptable toxic effects., [Main Outcomes and Measures] Adverse events were graded with the use of the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Tumor response was evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1., [Results] A total of 20 HIV-1–infected patients with advanced cancer were enrolled; 16 (80%) were male, the median (range) age was 54 (30-73) years, and 12 (60%) had progressed with previous cancer treatment lines. A median (range) of 4 (1-16) cycles of durvalumab were administered. Drug-related adverse events were observed in 50% of patients, and all were grade 1 and 2 (mainly diarrhea, asthenia, and arthromyalgia). Four of 16 response-evaluable patients (25%) had a partial response. Five patients (31%) had stable disease, including 4 with durable stable disease (disease control rate of 50%). CD4+ and CD8+ T-cell counts and plasma HIV-1 viremia remained stable throughout the study., [Conclusions and Relevance] Durvalumab treatment was feasible and safe in HIV-1–infected patients with cancer receiving combination antiretroviral therapy. HIV-1–infected patients on suppressive antiretroviral therapy with advanced cancer should have access to cancer immunotherapy treatments., [Trial Registration] ClinicalTrials.gov Identifier: NCT03094286.

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vacc

One of the main criticisms to DNA vaccines is the poor immunogenicity that they confer on occasions, at least in large animals. Confirming this theory, immunization with plasmid DNA encoding two African swine fever virus genes in frame (pCMV-PQ), failed in inducing detectable immune responses in pigs, while it was successful in mice. Aiming to improve the immune responses induced in swine, a new plasmid was constructed, encoding the viral genes fused in frame with a single chain variable fragment of an antibody specific for a swine leukocyte antigen II (pCMV-APCH1PQ). Our results clearly demonstrate that targeting antigens to antigen professional cells exponentially enhanced the immune response induced in pigs, albeit that the DNA vaccine was not able to confer protection against lethal viral challenge. Indeed, a viremia exacerbation was observed in each of the pigs that received the pCMV-APCH1PQ plasmid, this correlating with the presence of non-neutralizing antibodies and antigen-specific SLA II-restricted T-cells. The implications of our discoveries for the development of future vaccines against African swine fever virus and other swine pathogens are discussed. © 2011 Elsevier Ltd.

Classical swine fever (CSF) is a highly contagious viral infection affecting domestic and wild pigs. For classical swine fever virus (CSFV), immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection. Previously, we have also shown that immunization with DNA vaccine expressing glycoprotein E2 (DNA-E2) induced specific T helper cell responses in the absence of neutralizing antibodies. However, the role of T cell responses in protection against CSFV is largely unknown.Here we have extended these studies to deeply characterize the role of T cell responses by a DNA-E2 and their correlation with protection against CSFV infection. Thus, pigs vaccinated with the DNA vaccine induced a strong cellular immune response, characterized by the specific induction IFN-gamma expressing T cells after vaccination without any detectable levels of CSFV neutralizing antibodies. Constant levels of CSFV-specific IFN-gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed.Severe clinical signs of the disease, including high titers of viremia, pyrexia and virus spread to different organs, were recorded in the non-vaccinated challenged animals, in comparison to the vaccinated animals where only one animal showed mild clinical signs and a short peak of viremia. Lack of complete protection in this animal correlated with a delay on the induction of neutralizing antibodies, detectable only from day 11 post-CSFV challenge. Conversely, the rest of the pigs within the group developed neutralizing antibodies as early as at day two post-challenge, correlating with sterile protection. Finally, an inverse correlation seemed to exist between early induction of IFN-alpha and the protection observed, while IL-10 seemed to be differentially regulated in vaccinated and non-vac

Porcine circovirus type 2 (PCV2) vaccination has been recently included as a measure to control postweaning multisystemic wasting syndrome (PMWS) in the field. Aiming to obtain a more affordable vaccine to be extensively implemented in the field, a highly efficient non-fermentative expression platform based on Trichoplusia ni (T. ni) larvae was used to produce a baculovirus-derived recombinant PCV2 Cap protein (rCap) for vaccine purposes. Vaccination of pigs with rCap induced solid protection against PCV2 experimental infection, inhibiting both the viremia and the viral shedding very efficiently. The protection afforded by the rCap vaccine strongly correlated with the induction of specific humoral immune responses, even in the presence of PCV2-specific maternal immunity, although cellular responses also seemed to play a partial role. In summary, we have shown that rCap expressed in T. ni larvae could be a cost-effective PCV2 vaccine candidate to be tested under field conditions. © 2009 Elsevier Ltd.

In this study, a new in situ hybridisation (ISH) protocol has been developed to identify African swine fever virus (ASFV) genome in formalin-fixed, paraffin-embedded tissues. Different digoxigenin labelled ASFV-probes were tested, including single ASFV-specific oligonucleotides, an 18.5. kb restriction fragment from the viral genome and the entire ASFV genome. The latter showed the highest sensitivity in all tissues tested, independently of the virus used for challenge E75L or Ba71L. Although a similar ASFV genome distribution was observed, the number of ISH-positive cells was higher for Ba71L compared to E75L infected tissues. As expected, the monocyte-macrophage cell lineage was the main target cell for ASFV infection. Corresponding with the last stages of infection, ISH-positive signals were also found in other cell types, including endothelial cells, hepatocytes and neutrophils. Furthermore, two unexpected findings were also noticed the detection of a specific ISH-signal in lymphocytes and a tendency to find the signal in the nucleus of infected cells. In summary, the present findings demonstrate the utility of this new ISH protocol to study ASFV pathogenesis and its potential use as a diagnostic tool. © 2010 Elsevier B.V.

The main aim of the present study was to describe new methods for the identification of antibodies against the PCV2 capsid (Cap) and replicase (Rep) proteins in pig sera. Specifically, two new indirect enzyme-linked immunosorbent assays (ELISA) were developed based on recombinant PCV2 Cap (rCap) and Rep/Rep′ (rRep) proteins expressed in baculovirus and produced in Trichoplusia ni insect larvae. Both assays were validated by testing serum samples in a longitudinal study of 107 animals with different clinico-pathological features of PCV2 infection pigs with postweaning multisystemic wasting syndrome (PMWS), wasted pigs without a diagnosis of PMWS and healthy animals. Longitudinal antibody profiles indicated that healthy animals had significantly higher anti-Cap and anti-Rep antibody levels than the rest of the animal groups at 11 weeks of age. Moreover, PMWS affected pigs could be distinguished from the rest of the pig groups by their lower anti-Rep antibody levels at 11 weeks of age and at necropsy. The results demonstrate the potential of these two ELISAs for large-scale serological studies. This study represents the first longitudinal study of the induction of anti-Cap and anti-Rep antibodies in farms affected by PMWS, from 1 week of age until the occurrence of disease. © 2008 Elsevier B.V. All rights reserved.