Broad Protection against Invasive Fungal Disease from a Nanobody Targeting the Active Site of Fungal ß-1,3-Glucanosyltransferases.

Invasive fungal disease accounts for about 3.8 million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotype D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insight about the role of ß-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.

Molecular basis for bacterial N-glycosylation by a soluble HMW1C-like N-glycosyltransferase.

Soluble HMW1C-like N-glycosyltransferases (NGTs) catalyze the glycosylation of Asn residues in proteins, a process fundamental for bacterial autoaggregation, adhesion and pathogenicity. However, our understanding of their molecular mechanisms is hindered by the lack of structures of enzymatic complexes. Here, we report structures of binary and ternary NGT complexes of Aggregatibacter aphrophilus NGT (AaNGT), revealing an essential dyad of basic/acidic residues located in the N-terminal all α-domain (AAD) that intimately recognizes the Thr residue within the conserved motif Asn0-X+1-Ser/Thr+2. Poor substrates and inhibitors such as UDP-galactose and UDP-glucose mimetics adopt non-productive conformations, decreasing or impeding catalysis. QM/MM simulations rationalize these results, showing that AaNGT follows a SN2 reaction mechanism in which the acceptor asparagine uses its imidic form for catalysis and the UDP-glucose phosphate group acts as a general base. These findings provide key insights into the mechanism of NGTs and will facilitate the design of structure-based inhibitors to treat diseases caused by non-typeable H. influenzae or other Gram-negative bacteria.

A cross-sectional serosurvey of SARS-CoV-2 and co-infections in stray cats from the second wave to the sixth wave of COVID-19 outbreaks in Spain.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causative agent of Coronavirus Disease 2019 in humans. Among domestic animals, cats are more susceptible to SARS-CoV-2 than dogs. The detection of anti-SARS-CoV-2 antibodies in seemingly healthy cats and/or infected cats which are in close contact with infected humans has been described. The presence of animals that tested positive by serology or molecular techniques could represent a potential transmission pathway of SARS-CoV-2 that can spill over into urban wildlife. This study analyses the seroprevalence variation of SARS-CoV-2 in stray cats from different waves of outbreaks in a geographical area where previous seroepidemiological information of SARS-CoV-2 was available and investigate if SARS-CoV-2-seropositive cats were exposed to other co-infections causing an immunosuppressive status and/or a chronic disease that could lead to a SARS-CoV-2 susceptibility. For this purpose, a total of 254 stray cats from Zaragoza (Spain) were included. This analysis was carried out by the enzyme-linked immunosorbent assay using the receptor binding domain of Spike antigen and confirmed by serum virus neutralization assay. The presence of co-infections including Toxoplasma gondii, Leishmania infantum, Dirofilaria immitis, feline calicivirus, feline herpesvirus type 1, feline leukemia virus and feline immunodeficiency virus, was evaluated using different serological methods. A seropositivity of 1.57% was observed for SARS-CoV-2 including the presence of neutralizing antibodies in three cats. None of the seropositive to SARS-CoV-2 cats were positive to feline coronavirus, however, four SARS-CoV-2-seropositive cats were also seropositive to other pathogens such as L. infantum, D. immitis and FIV (n = 1), L. infantum and D. immitis (n = 1) and L. infantum alone (n = 1).Considering other pathogens, a seroprevalence of 16.54% was detected for L. infantum, 30.31% for D. immitis, 13.78%, for T. gondii, 83.86% for feline calicivirus, 42.52% for feline herpesvirus type 1, 3.15% for FeLV and 7.87% for FIV.Our findings suggest that the epidemiological role of stray cats in SARS-CoV-2 transmission is scarce, and there is no increase in seropositivity during the different waves of COVID-19 outbreaks in this group of animals. Further epidemiological surveillances are necessary to determine the risk that other animals might possess even though stray cats do not seem to play a role in transmission.

Preclinical Studies of Granulysin-Based Anti-MUC1-Tn Immunotoxins as a New Antitumoral Treatment.

Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosylated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.

Serological evidence of SARS-CoV-2 and co-infections in stray cats in Spain.

A new coronavirus known as SARS-CoV-2 emerged in Wuhan in 2019 and spread rapidly to the rest of the world causing the pandemic disease named coronavirus disease of 2019 (COVID-19). Little information is known about the impact this virus can cause upon domestic and stray animals. The potential impact of SARS-CoV-2 has become of great interest in cats due to transmission among domestic cats and the severe phenotypes described recently in a domestic cat. In this context, there is a public health warning that needs to be investigated in relation with the epidemiological role of this virus in stray cats. Consequently, in order to know the impact of the possible transmission chain, blood samples were obtained from 114 stray cats in the city of Zaragoza (Spain) and tested for SARS-CoV-2 and other selected pathogens susceptible to immunosuppression including Toxoplasma gondii, Leishmania infantum, feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) from January to October 2020. Four cats (3.51%), based on enzyme-linked immunosorbent assay (ELISA) using the receptor binding domain (RBD) of Spike antigen, were seroreactive to SARS-CoV-2. T. gondii, L. infantum, FeLV and FIV seroprevalence was 12.28%, 16.67%, 4.39% and 19.30%, respectively. Among seropositive cats to SARS-CoV-2, three cats were also seropositive to other pathogens including antibodies detected against T. gondii and FIV (n = 1); T. gondii (n = 1); and FIV and L. infantum (n = 1). The subjects giving positive for SARS-CoV-2 were captured in urban areas of the city in different months: January 2020 (2/4), February 2020 (1/4) and July 2020 (1/4). This study revealed, for the first time, the exposure of stray cats to SARS-CoV-2 in Spain and the existence of concomitant infections with other pathogens including T. gondii, L. infantum and FIV, suggesting that immunosuppressed animals might be especially susceptible to SARS-CoV-2 infection.

Absence of SARS-CoV-2 Antibodies in Natural Environment Exposure in Sheep in Close Contact with Humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the zoonotic causative agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic situation with millions of infected humans worldwide. Among domestic animals, there have been limited studies regarding the transmissibility and exposure to the infection in natural conditions. Some animals are exposed and/or susceptible to SARS-CoV-2 infection, such as cats, ferrets and dogs. By contrast, there is no information about the susceptibility of ruminants to SARS-CoV-2. This study tested the antibody response in 90 ovine pre-pandemic serum samples and 336 sheep serum samples from the pandemic period (June 2020 to March 2021). In both cases, the animals were in close contact with a veterinary student community composed of more than 700 members. None of the serum samples analyzed was seroreactive based on an enzyme-linked immunosorbent assay (ELISA) using the receptor-binding domain (RBD) of the spike antigen. In this sense, no statistical difference was observed compared to the pre-pandemic sheep. Our results suggest that it seems unlikely that sheep could play a relevant role in the epidemiology of SARS-CoV-2 infection. This is the first study to report the absence of evidence of sheep exposure to SARS-CoV-2 in natural conditions.

Determination of the Concentration of IgG against the Spike Receptor-Binding Domain That Predicts the Viral Neutralizing Activity of Convalescent Plasma and Serum against SARS-CoV-2.

Several hundred millions of people have been diagnosed of coronavirus disease 2019 (COVID-19), causing millions of deaths and a high socioeconomic burden. SARS-CoV-2, the causative agent of COVID-19, induces both specific T- and B-cell responses, being antibodies against the virus detected a few days after infection. Passive immunization with hyperimmune plasma from convalescent patients has been proposed as a potentially useful treatment for COVID-19. Using an in-house quantitative ELISA test, we found that plasma from 177 convalescent donors contained IgG antibodies specific to the spike receptor-binding domain (RBD) of SARS-CoV-2, although at very different concentrations which correlated with previous disease severity and gender. Anti-RBD IgG plasma concentrations significantly correlated with the plasma viral neutralizing activity (VN) against SARS-CoV-2 in vitro. Similar results were found using an independent cohort of serum from 168 convalescent health workers. These results validate an in-house RBD IgG ELISA test in a large cohort of COVID-19 convalescent patients and indicate that plasma from all convalescent donors does not contain a high enough amount of anti-SARS-CoV-2-RBD neutralizing IgG to prevent SARS-CoV-2 infection in vitro. The use of quantitative anti-RBD IgG detection systems might help to predict the efficacy of the passive immunization using plasma from patients recovered from SARS-CoV-2.

Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody.

The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.

Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology.

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris. In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn+ cell lines was higher than that of granulysin alone.