Austral University of Chile(UACh), Chile

History of the Austral University of Chile(UACh)

 The Austral University of Chile, UACh, dates back to 1942 when a society called “Society of the Friends of the Art) was formed in the city of Valdivia to promote culture. One of the society's main goals was the establishment of a university in the city.
Today, the Austral University of Chile account for 16.000 undergraduate students, 1.500 postgraduates’ students and 1.900 academic and support staff. UACh works in alliance with the e majors Hospitals in the south of the country in research areas and academic matters.

Campus Isla Teja UACh

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The main campus of the UACh on the Teja Island, occupies the whole northwest of Isla Teja in Valdivia. Campus Isla Teja is the home of the UACh's administration, Cinema Club, Botanical Garden and most Research Departments. The Botanical Garden is a recreational area as well as a place of study with a total of around 950 plant species growing in its 12 ha. Most plants in the Botanical Garden belong to Valdivian temperate rain forest but there are others with exotic origins. The Cau-Cau River flows through the northern parts of the botanical garden and allows a section of wetland plant species included in the garden.
The engineering department operates at the Campus Miraflores, located alongside the Valdivia River south of Valdivia's Plaza de Armas. A third campus exists in Puerto Montt, where medical sciences, speech and language pathology, business administration, law, IT, among other undergrad careers are offered. The Puerto Montt Campus also has a strong graduate program on aquaculture. The UACh has - apart from the campuses - several properties spread through the Los Ríos and the Los Lagos Regions such as Parque Arboretum in northern Isla Teja, the Calfuco field station at the coast near Niebla and the forests of San Pablo de Tregua in the Andean foothills.

Platform for generation of single-domain antibody (sdAb) from Camelids- Available for KOICID

In nature, there are a few exceptions of functional antibodies lacking the light chains, known as single domain antibodies (SdAb). They are derivates of bona fide IgG and occur in the entire Camilidae family, nurse shark, wobbegong, and perhaps spotted ratfish The family of camelids comprises camels, dromedars, llamas, vicuñas, guanaco and alpacas The antigen-binding fragment of an SdAb contains a single variable VHH domain consisting of 3 hypervariable regions. When isolated, the VHH domain is also known as SdAb. The target-specific SdAbs derived from SdAb s of camelids are generally rapidly obtained briefly after immunization with the target protein plus adjuvant. The solved structure of the SdAbs reveals how the hypervariable regions are projected in loops outside of the core structure. In order to isolate the genomic sequences of the target specific SdAbs, first it is necessary to isolate the peripheral B-lymphocytes to isolate total RNA, and further to perform a cDNA preparation to finally amplify the SdAb region from gene V of the repertoire. The fragment encoding the SdAb in gene V is as short as 360nt long and from 50mL of blood it is possible to obtain up to 1x106 single SdAb clones. The SdAb sequences are cloned in a phage display vector, thus, after transformation in the competent phage replication strain, each single transfected bacterium will produce ~100 phages increasing the possibilities of identifying less abundant SdAb clones. The phage display technology allows the expression of the SdAb on the surfaces of bacteriophages and, therefore, it is possible to enrich the phage expressing the SdAb of interest by affinity purification. This process is known as panning. The final isolated SdAbs are recombinantly expressed in bacteria and their binding abilities can be characterized by ELISA and quantitative biochemical parameters such as ITC. In addition to the directly targeted immunization, it is also possible to use very large libraries of phage display SdAbs (1x109) to find binding SdAbs by a stochastically approach.  SdAbs are produced then in a renewable and economical manner. Further advantage of SdAbs are: the small size, they can be humanized, their stable structure and behaviour in aqueous solutions, the specific and high affinity binding to a single target protein and the natural production by camelids. Therefore, SdAbs are the best tools available today for affinity-based diagnostic and therapies.

Alpacas for SdAbs generation at the Austral University of Chile

Here, we summarised the advantages and uses of SdAbs in detail.
 

Advantages of SdAbs

Stability:SdAbs are small and compact polypeptides and they are often expressed in the periplasm of bacteria. They have been shown to be very stable at high temperatures starting at 6°C compared to human VH and they are also resistant to denaturing chemical agents.  Furthermore, molecular engineering of the SdAb structure has shown that the stability increases by introducing a cysteine at position 54 and 78 to form an extra disulphide bond. Interestingly, the resulting super stable SdAb is also more resistant to proteases such as pepsin or chymotrypsin.

Immune invisibility:SdAbs can be used as therapeutic bullets against tumours, pathogens and chronical diseases, however as foreign proteins, they could trigger an immune response themselves. Fortunately, the small size, rapid clearance from the blood and high homology to the human variable region of the heavy chain VH makes them very little immunogenic. Even more, only 14 amino acids differ between SdAbs and the human VH, the substitution of 12 camelid amino acids for human amino acids has been used to humanise the camelid SdAbs and make them even more save for therapies.

Accessibility:SdAbs are strict monomers, their affinity for substrates depend of the projection of the three hypervariable loops. In consequence, SdAbs tent to interact to cavities of the spatial structure of polypeptides, but not efficiently to peptides. For instance, several identified SdAbs directly block active sites of enzymes. In addition, the FC5 SdAb can even cross the blood-brain barrier by transcytosis and form partly bispecific antibodies used for therapeutic approaches. Finally, the molecular accessibility impacts the access to macromolecular complexes.

Use of SdAbs

​Structural studies:When a SdAb binds to a three-dimensional structure, some properties of the targeted protein change. One of the common features is that SdAbs limit the conformational changes of the target protein promoting a singular conformation stage. Remarkably, these phenomena impact positively the chances of crystallization, therefore, SdAbs have been used as helpers to facilitate the crystallization of proteins.

Diagnostic: SdAbs are a superior tool for diagnostic. Their unlimited capacity of production in vitro makes SdAbs more reliable than conventional antibodies and independent of batch preparation or animal serum limitations. SdAbs can be produced as a protein fusion with reporter peptides or proteins for direct staining or visualisation of the SdAbs, those include affinity tags (Flag, HA, V5 and cMyc), fluorescent proteins (GFP, RFP, etc) and enzymes for colorimetric measurements such as horseradish peroxidase (HRP). For instance, it is possible to improve ELISA assays using SdAbs for either specific immobilisation or for detection using a specific SdAb coupled to HRP.  SdAbs fulfil most of the requisites of an ideal probe for successful molecular imaging. Several imaging techniques such as SPECT, PET, optical imaging and ultrasound have been successfully employed for molecular imaging using SdAbs in vivo. In the case of SPECT, specific targeted SdAbs are coupled to a source of γ-irradiation and delivered systemically, subsequently, the SdAbs are detected in a full body scanner. The strategy of PET imaging is similar to SPECT but using positron-emitting radionuclide-labelled SdAbs instead. These imaging technologies have been used for several tumour diagnostics in animal models, for example using an anti-EGFR SdAb it is possible to detect human epidermoid carcinoma and human prostate carcinoma. In another extraordinary case, they have used a combination of SdAbs targeting the carbonic anhydrase IX (CAIX) and human epidermal growth factor receptor 2 (HER2) proteins conjugated to different fluorophores. Here, the authors conclude that the simultaneous determination of the expression status of multiple, clinically relevant tumour markers could lead to a better detection of the primary tumour and its metastases.

Therapies: Several SdAbs have been developed in the context of different experimental therapeutic applications against different viruses: HIV, Hepatitis B virus, influenza virus, Respiratory Syncytial virus, Rabies virus, FMDV, Poliovirus, Rotavirus, and PERVs. Remarkably, SdAbs can be used to neutralize HIV infection; cell to cell spread has been inhibited using HIV isolated from patients. Due to the low immunoreactivity of SdAbs, they can be injected into patients with very little or no secondary effects. In order to make them more efficient and specific, SdAbs can be linked to produce bivalent, multivalent, and/or multispecific SdAbs, or combined with other SdAbs or circulating proteins such as albumin to increase their turn over and therapeutic effectiveness. Rabies virus causes lethal brain infection in people. Soon after exposure anti-rabies prophylaxis is provided with plasma-derived immunoglobulins and vaccines. Often, this occurs directly after the attack of an animal which could be infected. Anti-rabies SdAbs are able to significantly prolong survival or even completely heal the disease in animal models. Respiratory Syncytial Virus, RSV, is one of the major causes of hospitalization in children, every year more than 1.9 millions of children <1 years are infected and there are over 0.3 million children <5 years in hospitalization. No current therapy is available against RSV. However, a trivalent SdAb-based therapy is in clinical studies phase II. The absolute novelty of the RSV therapy developed by Ablynx, ALX-0171, is the direct neutralization of the virus in the lung of infected experimentation animals. The SdAb is administered by inhalation through the airway by nebulisation. The therapy reduced the virus titre by 10.000 times. SdAbs are also used for immunotherapies against cancers. There is an extraordinary potential of the SdAb technology in a way the molecular biotechnology is able to borrow elements from the camelid immune system to fight human diseases. In the laboratory for camelid biotechnology we already have 3 alpacas sera positive against the nucleoprotein of the Hantavirus Andes, further isolation of sdAbs is ongoing

Other research Areas UACh

The UACh is one of the leading universities of Chile in matters of scientific research, the university covers several areas of the knowledge as:

Dairy technology and science: The University’s Centro Tecnológico de la Leche (lit. Milk Technology Centre) has been accredited by the scholar Paulina Rytkönen for being behind the higher dairy productivity in Los Ríos Region when compared with the adjacent Los Lagos Region.

Dendrochronology: Scientists at the Dendrochronology Laboratory of UACh have set up the longest dendrochronology in the Southern Hemisphere going back 5666 years. Researchers have also reconstructed the temperatures of the last 3622 years with tree rings from Fitzroya and have used a dendrochronology network of Lenga Beech, spanning from Talca to Navarino Island, to investigate global warming in the recent decades.

Palaeontology: Palaeontological studies in Southern Chile have involved several UACh academics, amongst them the geoarchaeologist Mario Pino who participated in the excavation of the Quaternary fossil site of Pilauco Bajo in Osorno where the remains of a species of Gomphotherium were discovered. Two other Gomphotherium fossil sites in Los Ríos Region are planned to be excavated by university scientists in the future.

Virology: Researchers from the department of Medicine and department of Science led a series of joint medical, viral and zoological projects on the transmittance of Hantavirus between rodents and humans. Hantavirus was first detected in Chile in 1995 but it is believed to have been present much longer before. Hanta antigens were found on the salivary glands of deceased persons. UACh academics working at the Laboratory of Virology had contributed to diminish the initial mortality of Pulmonary Hantavirus in Los Ríos Region below the national average of 37%.

Microbiology: The faculty of sciences, the faculty of medicine and the faculty of veterinarian medicine account with microbiologist teams advocated to bacterial, parasites and Mycology. Strong association have been built with European researchers around the one health concept attempting the better understanding of microorganism, zoonosis and the risk associated with Climate Change and global warming.

Epidemiology: Strong efforts have been done after the Q Fever caused by Coxiella burnetii outbreak of 2017-2018. A direct funding from the health minister currently studies possible local vectors of the diseases in collaboration with the Friedrich-Loeffler Institute (FLI) in Germany. The research initiative is developed by Dr. Carla Rosenfeld and Dr. Ananda Muller and also covers the detection of rickettsia’s and Bartonella spp and the development of new technologies for detection.

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STAFF

​Professor Dr. Alejandro Rojas Fernandez

HOMEPAGE

www.uach.cl/
www.medicina.uach.cl/
http://biotechnology-lab.com/
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