Antigen identification and high-throughput interaction mapping by reprogramming viral entry.

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.

V-CARMA: A tool for the detection and modification of antigen-specific T cells.

T cells promote our body's ability to battle cancers and infectious diseases but can act pathologically in autoimmunity. The recognition of peptides presented by major histocompatibility complex (pMHC) molecules by T cell receptors (TCRs) enables T cell-mediated responses. To modify disease-relevant T cells, new tools to genetically modify T cells and decode their antigen recognition are needed. Here, we present an approach using viruses pseudotyped with peptides loaded on MHC called V-CARMA (Viral ChimAeric Receptor MHC-Antigen) to specifically target T cells expressing cognate TCRs for antigen discovery and T cell engineering. We show that lentiviruses displaying antigens on human leukocyte antigen (HLA) class I and class II molecules can robustly infect CD8+ and CD4+ T cells expressing cognate TCRs, respectively. The infection rates of the pseudotyped lentiviruses (PLVs) are correlated with the binding affinity of the TCR to its cognate antigen. Furthermore, peptide-HLA pseudotyped lentivirus V-CARMA constructs can identify target cells from a mixed T cell population, suppress PD-1 expression on CD8+ T cells via PDCD1 shRNA delivery, and induce apoptosis in autoreactive CD4+ T cells. Thus, V-CARMA is a versatile tool for TCR ligand identification and selective T cell manipulation.

Less phagocytosis of viral vectors by tethering with CD47 ectodomain.

Many viral vectors, which are effective when administrated in situ, lack efficacy when delivered intravenously. The key reason for this is the rapid clearance of the viruses from the blood circulation via the immune system before they reach target sites. Therefore, avoiding their clearance by the immune system is essential. In this study, lentiviral vectors were tethered with the ectodomain of self-marker protein CD47 to suppress phagocytosis via interacting with SIRPα on the outer membrane of macrophage cells. CD47 ectodomain and core-streptavidin fusion gene (CD47ED-coreSA) was constructed into pET-30a(+) plasmid and transformed into Lemo21 (DE3) competent E. coli cells. The expressed CD47ED-coreSA chimeric protein was purified by cobalt-nitrilotriacetate affinity column and characterized by SDS-PAGE and western blot. The purified chimeric protein was anchored on biotinylated lentivirus via biotin-streptavidin binding. The CD47ED-capped lentiviruses encoding GFP were used to infect J774A.1 macrophage cells to assess the impact on phagocytosis. Our results showed that the overexpressed CD47ED-coreSA chimeric protein was purified and bound on the surface of biotinylated lentivirus which was confirmed via immunoblotting assay. The process to produce biotinylated lentivirus did not affect native viral infectivity. It was shown that the level of GFP expression in J774A.1 macrophages transduced with CD47ED-lentiviruses was threefold lower in comparison to control lentiviruses, indicating an antiphagocytic effect triggered by the interaction of CD47ED and SIRPα. Through the test of blocking antibodies against CD47ED and/or SIRPα, it was confirmed that the phagocytosis inhibition was mediated through the CD47ED-SIRPα axis signaling. In conclusion, surface immobilization of CD47ED on lentiviral vectors inhibits their phagocytosis by macrophages. The chimeric protein of CD47 ectodomain and core-streptavidin is effective in mediating the surface binding and endowing the lentiviral nanoparticles with the antiphagocytic property.

Serological, Molecular and Culture-Based Diagnosis of Lentiviral Infections in Small Ruminants.

Small ruminant lentiviruses (SRLVs) infections lead to chronic diseases and remarkable economic losses undermining health and welfare of animals and the sustainability of farms. Early and definite diagnosis of SRLVs infections is the cornerstone for any control and eradication efforts; however, a "gold standard" test and/or diagnostic protocols with extensive applicability have yet to be developed. The main challenges preventing the development of a universally accepted diagnostic tool with sufficient sensitivity, specificity, and accuracy to be integrated in SRLVs control programs are the genetic variability of SRLVs associated with mutations, recombination, and cross-species transmission and the peculiarities of small ruminants' humoral immune response regarding late seroconversion, as well as intermittent and epitope-specific antibody production. The objectives of this review paper were to summarize the available serological and molecular assays for the diagnosis of SRLVs, to highlight their diagnostic performance emphasizing on advantages and drawbacks of their application, and to discuss current and future perspectives, challenges, limitations and impacts regarding the development of reliable and efficient tools for the diagnosis of SRLVs infections.

Development of Lentiviral Vectors Pseudotyped With Influenza B Hemagglutinins: Application in Vaccine Immunogenicity, mAb Potency, and Sero-Surveillance Studies.

Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research.

Integrase-derived peptides together with CD24-targeted lentiviral particles inhibit the growth of CD24 expressing cancer cells.

The integration of viral DNA into the host genome is mediated by viral integrase, resulting in the accumulation of double-strand breaks. Integrase-derived peptides (INS and INR) increase the number of integration events, leading to escalated genomic instability that induces apoptosis. CD24 is a surface protein expressed mostly in cancer cells and is very rarely found in normal cells. Here, we propose a novel targeted cancer therapeutic platform based on the lentiviral integrase, stimulated by integrase-derived peptides, that are specifically delivered to cancerous cells via CD24 antigen-antibody targeting. INS and INR were synthesized and humanized and anti-CD24 antibodies were fused to the lentivirus envelope. The activity, permeability, stability, solubility, and toxicity of these components were analyzed. Cell death was measured by fluorescent microscopy and enzymatic assays and potency were tested in vitro and in vivo. Lentivirus particles, containing non-functional DNA led to massive cell death (40-70%). Raltegravir, an antiretroviral drug, inhibited the induction of apoptosis. In vivo, single and repeated administrations of INS/INR were well tolerated without any adverse effects. Tumor development in nude mice was significantly inhibited (by 50%) as compared to the vehicle arm. In summary, a novel and generic therapeutic platform for selective cancer cell eradication with excellent efficacy and safety are presented.

Anti-CD19 CAR T cells potently redirected to kill solid tumor cells.

Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.

SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus.

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

Intranasal vaccination with a lentiviral vector protects against SARS-CoV-2 in preclinical animal models.

To develop a vaccine candidate against coronavirus disease 2019 (COVID-19), we generated a lentiviral vector (LV) eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, confers only partial protection despite high levels of serum neutralizing activity. However, eliciting an immune response in the respiratory tract through an intranasal boost results in a >3 log10 decrease in the lung viral loads and reduces local inflammation. Moreover, both integrative and non-integrative LV platforms display strong vaccine efficacy and inhibit lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and closely mirror human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of LV-based vaccination against SARS-CoV-2 and designate intranasal immunization as a powerful approach against COVID-19.

Evaluation of Nonviral piggyBac and lentiviral Vector in Functions of CD19chimeric Antigen Receptor T Cells and Their Antitumor Activity for CD19+ Tumor Cells.

Nonviral transposon piggyBac (PB) and lentiviral (LV) vectors have been used to deliver chimeric antigen receptor (CAR) to T cells. To understand the differences in the effects of PB and LV on CAR T-cell functions, a CAR targeting CD19 was cloned into PB and LV vectors, and the resulting pbCAR and lvCAR were delivered to T cells to generate CD19pbCAR and CD19lvCAR T cells. Both CD19CAR T-cell types were strongly cytotoxic and secreted high IFN-γ levels when incubated with Raji cells. TNF-α increased in CD19pbCAR T cells, whereas IL-10 increased in CD19lvCAR T cells. CD19pbCAR and CD19lvCAR T cells showed similar strong anti-tumor activity in Raji cell-induced mouse models, slightly reducing mouse weight while enhancing mouse survival. High, but not low or moderate, concentrations of CD19pbCAR T cells significantly inhibited Raji cell-induced tumor growth in vivo. These CD19pbCAR T cells were distributed mostly in mesenteric lymph nodes, bone marrow of the femur, spleen, kidneys, and lungs, specifically accumulating at CD19-rich sites and CD19-positive tumors, with CAR copy number being increased on day 7. These results indicate that pbCAR has its specific activities and functions in pbCAR T cells, making it a valuable tool for CAR T-cell immunotherapy.

Serotyping versus genotyping in infected sheep and goats with small ruminant lentiviruses.

Despite SRLV infection being endemic in Mexico, there is little information regarding which genotypes are present. We compared serotyping and PCR-sequencing results from sheep and goats infected with SRLV. We separated plasma and peripheral blood leukocytes (PBL) from 1940 blood samples from sheep and goats from 12 states across Mexico. To detect SRLV infection, we tested plasma samples using two commercial ELISA kits (VMRD and Eradikit SRLV Screening). Then, we serotyped the infecting virus (A/ B) using Eradikit SRLV Genotyping. PBL DNA was used to detect the proviral genome via PCR. Positive amplicons were sequenced to identify viral genotypes using a phylogenetic analysis. Also, we analysed for residues differences in the sequences of a capsid epitope between genotypes. The serological results indicated a higher detection of seropositive animals using the VMRD ELISA compared to Eradikit, with 21 % and 15.3 % more in sheep and goats respectively. Only 25.7 % of the ELISA serotyping results matched those from PCR-sequencing. PCR-sequencing was able to identify genotype A, B and coinfections in animals classified as indeterminate by the ELISA test. This lack of sensitivity may be related to the lack of epitopes from the matrix and transmembrane peptides used by ELISA screening. Sequences analysis revealed that SRLVs found in sheep cluster with genetic subtypes A2 and B1, while those in goats cluster with subtypes A1 and B1. Serotyping did not prove to be an adequate method for predicting the viral genotype (A and / or B) in infections caused by SRLV.

Persistent lentivirus infection induces early myeloid suppressor cells expansion to subvert protective memory CD8 T cell response✰,✰✰.

BACKGROUND: Memory CD8+T cell responses play an essential role in protection against persistent infection. However, HIV-1 evades vaccine-induced memory CD8+T cell response by mechanisms that are not fully understood. METHODS: We analyzed the temporal dynamics of CD8+T cell recall activity and function during EcoHIV infection in a potent PD1-based vaccine immunized immunocompetent mice. FINDINGS: Upon intraperitoneal EcoHIV infection, high levels of HIV-1 GAG-specific CD8+T lymphocytes recall response reduced EcoHIV-infected cells significantly. However, this protective effect diminished quickly after seven days, followed by a rapid reduction of GAG-specific CD8+T cell number and activity, and viral persistence. Mechanistically, EcoHIV activated dendritic cells (DCs) and myeloid cells. Myeloid cells were infected and rapidly expanded, exhibiting elevated PD-L1/-L2 expression and T cell suppressive function before day 7, and were resistant to CD8+T cell-mediated apoptosis. Depletion of myeloid-derived suppressor cells (MDSCs) reduced EcoHIV infection and boosted T cell responses. INTERPRETATION: This study provides an overview of the temporal interplay of persistent virus, DCs, MDSCs and antigen-specific CD8+T cells during acute infection. We identify MDSCs as critical gatekeepers that restrain antiviral T cell memory responses, and highlight MDSCs as an important target for developing effective vaccines against chronic human infections. FUNDING: Hong Kong Research Grant Council (T11-709/18-N, HKU5/CRF/13G), General Research Fund (17122915 and 17114114), Hong Kong Health and Medical Research Fund (11100752, 14130582, 16150662), Grant RGC-ANR A-HKU709/14, the San-Ming Project of Medicine (SZSM201512029), University Development Fund of the University of Hong Kong and Li Ka Shing Faculty of Medicine Matching Fund to HKU AIDS Institute.

Structural Insights into APOBEC3-Mediated Lentiviral Restriction.

Mammals have developed clever adaptive and innate immune defense mechanisms to protect against invading bacterial and viral pathogens. Human innate immunity is continuously evolving to expand the repertoire of restriction factors and one such family of intrinsic restriction factors is the APOBEC3 (A3) family of cytidine deaminases. The coordinated expression of seven members of the A3 family of cytidine deaminases provides intrinsic immunity against numerous foreign infectious agents and protects the host from exogenous retroviruses and endogenous retroelements. Four members of the A3 proteins-A3G, A3F, A3H, and A3D-restrict HIV-1 in the absence of virion infectivity factor (Vif); their incorporation into progeny virions is a prerequisite for cytidine deaminase-dependent and -independent activities that inhibit viral replication in the host target cell. HIV-1 encodes Vif, an accessory protein that antagonizes A3 proteins by targeting them for polyubiquitination and subsequent proteasomal degradation in the virus producing cells. In this review, we summarize our current understanding of the role of human A3 proteins as barriers against HIV-1 infection, how Vif overcomes their antiviral activity, and highlight recent structural and functional insights into A3-mediated restriction of lentiviruses.

(Non-)Sense of Milk Testing in Small Ruminant Lentivirus Control Programs in Goats. Comparative Analysis of Antibody Detection and Molecular Diagnosis in Blood and Milk.

Small ruminant lentivirus (SRLV) control programs are mainly based on diagnostic tests performed on blood samples collected from sheep and goats. Since blood sampling is costly and stressful for the animals, we evaluated whether milk could be used as an inexpensive and easily collectable matrix for SRLV detection. We therefore compared SRLV detection via two commercial enzyme-linked immunosorbent assays (ELISAs) and quantitative polymerase chain reaction (qPCR) in blood and corresponding milk samples from 321 goats originating from eight different SRLV-infected farms in Flanders (Belgium). The IDscreen® ELISA had a better relative sensitivity (97% vs 93%) and specificity (100% and 97%) than the Elitest® ELISA for SRLV-specific antibody detection in milk compared to serum. The higher sensitivity correlates with a 10-fold higher analytical sensitivity of the IDscreen® test. In contrast to the overall good ELISA results, qPCR on milk cell pellets lacked sensitivity (81%) and specificity (88%), compared to molecular detection in blood leucocyte pellets. Our results show that serology is more suitable than qPCR for SRLV diagnosis, and that milk may represent an interesting matrix for a preliminary evaluation of a herd's infection status. Serum remains however the sample of choice for control programs where it is important to identify positive animals with the highest sensitivity.

Use of lentiviral pseudotypes as an alternative to reassortant or Triton X-100-treated wild-type Influenza viruses in the neuraminidase inhibition enzyme-linked lectin assay.

BACKGROUND: Formulation of neuraminidase (NA) within influenza vaccines is gaining importance in light of recent human studies. The enzyme-linked lectin assay (ELLA) is considered a reliable assay to evaluate human anti-NA antibodies. OBJECTIVES: To overcome interference by hemagglutinin (HA)-specific antibodies and detect neuraminidase inhibitory (NI) antibodies only, two different sources of antigen have been studied in ELLA: reassortant viruses with a mismatched avian origin-HA or Triton X-100 (Tx)-treated wild-type viruses. Pseudotypes or pseudovirus (PV), characterized by a lentivirus core bearing human influenza NA and avian influenza HA, were investigated as an alternative source of antigen and compared to HA-mismatched and Tx-treated viruses, since represent a safer product to be handled. METHODS: Two independent panels of sera were analyzed by ELLA to evaluate the anti-NA response against N1 (A/California/07/2009 (H1N1pdm)) and N2 (A/Hong Kong/4801/2014 (H3N2)). The NA inhibition (NI) antibody titers measured as either the 50% end point or 50% inhibitory concentration (IC50 ) were compared for every source of antigen. RESULTS: The ELLA assay performed well with all three sources of antigen. NI titers measured using each antigen type correlated well when reported either as end point titers or as the IC50 . CONCLUSIONS: This study suggests that HA-mismatched whole virus, Triton-treated wild-type virus or PV can be used to measure NI antibody titers of human sera, but further comparability/validation assays should be performed to assess statistical differences. The data support the use of PV as an attractive alternative source of antigen and justify further investigation to improve stability of this antigen source.

First-in-Class, First-in-Human Study Evaluating LV305, a Dendritic-Cell Tropic Lentiviral Vector, in Sarcoma and Other Solid Tumors Expressing NY-ESO-1.

PURPOSE: LV305 is a modified, third-generation, nonreplicating, integration-deficient lentivirus-based vector designed to selectively transduce dendritic cells in vivo. LV305 induces expression of the New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1) cancer testis antigen in dendritic cells, promoting immune responses against NY-ESO-1-expressing tumors. This phase I study evaluated the safety, immunogenicity, and preliminary efficacy of LV305 in patients with sarcoma or other solid tumors. PATIENTS AND METHODS: Adults with previously treated, advanced, NY-ESO-1-positive solid tumors and limited tumor burden were eligible. LV305 was administered every 3 weeks by intradermal injection in four dose cohorts (Cohort 1: 108 vector genomes (vg) x 3 doses; Cohorts 1A, 2, and 3: 108 vg, 109 vg, 1010 vg x 4 doses). RESULTS: Thirty-nine patients were enrolled: 3 patients each in Cohorts 1, 1A, and 2, and 30 patients in Cohort 3. No dose-limiting toxicities were observed. Tumor types included sarcoma (n = 24), ovarian (n = 8), melanoma (n = 6), and lung cancer (n = 1). All treatment-related adverse events were grade 1 or 2. Common treatment-related adverse events were fatigue (49%), injection site reactions (46%), and myalgia (21%). The disease control rate was 56.4% in all patients and 62.5% in sarcoma patients. One patient with synovial sarcoma achieved a partial response lasting >36 months. Anti-NY-ESO-1-specific CD4+ and/or CD8+ T cells were induced in 57% of evaluable sarcoma patients. Induction of an anti-NY-ESO-1 immune response was associated with improved 1-year survival in an exploratory analysis. CONCLUSIONS: This first-in-class, first-in-human study of LV305 demonstrated a favorable safety profile, induction of antigen-specific responses, and potential clinical activity in patients with advanced cancer.

Intertwined: SAMHD1 cellular functions, restriction, and viral evasion strategies.

SAMHD1 was initially described for its ability to efficiently restrict HIV-1 replication in myeloid cells and resting CD4+ T cells. However, a growing body of evidence suggests that SAMHD1-mediated restriction is by far not limited to lentiviruses, but seems to be a general concept that applies to most retroviruses and at least a number of DNA viruses. SAMHD1 anti-viral activity was long believed to be solely due to its ability to deplete cellular dNTPs by enzymatic degradation. However, since its discovery, several new functions have been attributed to SAMHD1. It has been demonstrated to bind nucleic acids, to modulate innate immunity, as well as to participate in the DNA damage response and resolution of stalled replication forks. Consequently, it is likely that SAMHD1-mediated anti-viral activity is not or not exclusively mediated through its dNTPase activity. Therefore, in this review, we summarize current knowledge on SAMHD1 cellular functions and systematically discuss how these functions could contribute to the restriction of a broad range of viruses besides retroviruses: herpesviruses, poxviruses and hepatitis B virus. Furthermore, we aim to highlight different ways how viruses counteract SAMHD1-mediated restriction to bypass the SAMHD1-mediated block to viral infection.

Modulation of immune responses in lentiviral vector-mediated gene transfer.

Lentiviral vectors (LV) are widely used vehicles for gene transfer and therapy in pre-clinical animal models and clinical trials with promising safety and efficacy results. However, host immune responses against vector- and/or transgene-derived antigens remain a major obstacle to the success and broad applicability of gene therapy. Here we review the innate and adaptive immunological barriers to successful gene therapy, both in the context of ex vivo and in vivo LV gene therapy, mostly concerning systemic LV delivery and discuss possible means to overcome them, including vector design and production and immune modulatory strategies.

Small ruminant lentiviruses in goats in southern Italy: Serological evidence, risk factors and implementation of control programs.

Small ruminant lentiviruses (SRLVs) can drastically affect milk production in goat flocks and only an early detection can control and prevent their spread. Since SRLVs are responsible for persistent infections, antibody screening is the most valuable tool to identify infected animals. ELISA is recommended as the election test both for its sensitivity and for its ability to detect low antibody titers, thus identifying infected animals earlier than agar gel immunodiffusion (AGID). In the present study, an investigation was conducted to assess the SRLV seroprevalence in goat flocks in southern Italy and a transversal comparative study was carried out through the analysis of the possible risk factors influencing SRLV spread. A total of 4800 sera from 1060 flocks were analyzed and overall seroprevalences of 18,64% and 51,69% at animal and herd levels, respectively, were observed. Both the region and the herd production systems were able to affect seroprevalence, differently from the herd size, probably because the mean number of goats per herd is low and the semi-intensive management is similar regardless of the dimensional class of each herd. In particular, meat producing herds showed the higher seroprevalence, as a result of the poor sanitation and low animal monitoring in comparison to milk producing herds, where animals are managed twice daily and the relationship between dams and kids is checked to guarantee an adequate quantitative/qualitative milk yield. In the absence of vaccines or effective treatments, health preventive management and seroepidemiological investigations are the only successful approach to restrict SRLV spread as observed in countries were official/voluntary control programs are carried out.

Development of Inducible CD19-CAR T Cells with a Tet-On System for Controlled Activity and Enhanced Clinical Safety.

The tetracycline regulatory system has been widely used to control the transgene expression. With this powerful tool, it might be possible to effectively control the functional activity of chimeric antigen receptor (CAR) T cells and manage the severe side effects after infusion. In this study, we developed novel inducible CD19CAR (iCAR19) T cells by incorporating a one-vector Tet-on system into the CD19CAR construct. The iCAR19 T cells showed dox-dependent cell proliferation, cytokine production, CAR expression, and strong CD19-specific cytotoxicity. After 48 h of dox induction, the relative CAR expression of induced cells was five times greater than that of uninduced cells. Twenty-four hours after dox removal, CAR expression significantly decreased by more than 60%. In cytotoxicity assays, dox-treated cells induced significantly higher specific lysis against target cells. These results suggested that the activity of iCAR19 T cells was successfully controlled by our Tet-on system, offering an enhanced safety profile while maintaining a robust anti-tumor effect. Besides, all manufacture processes of the lentiviral vectors and the T cells were conducted according to the Good Manufacturing Practice (GMP) standards for subsequent clinical translation.