Production of Recombinant Zika Virus Envelope Protein by Airlift Bioreactor as a New Subunit Vaccine Platform.

The Zika Virus (ZIKV) is an emerging arbovirus of great public health concern, particularly in the Americas after its last outbreak in 2015. There are still major challenges regarding disease control, and there is no ZIKV vaccine currently approved for human use. Among many different vaccine platforms currently under study, the recombinant envelope protein from Zika Virus (rEZIKV) constitutes an alternative option for vaccine development and has great potential for monitoring ZIKV infection and antibody response. This study describes a method to obtain a bioactive and functional rEZIKV using an E. coli expression system, with the aid of a 5-L airlift bioreactor and following an automated fast protein liquid chromatography (FPLC) protocol, capable of obtaining high yields of approximately 20 mg of recombinant protein per liter of bacterium cultures. The purified rEZIKV presented preserved antigenicity and immunogenicity. Our results show that the use of an airlift bioreactor for the production of rEZIKV is ideal for establishing protocols and further research on ZIKV vaccines bioprocess, representing a promising system for the production of a ZIKV envelope recombinant protein-based vaccine candidate.

RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

Chitosan: Applications in Drug Delivery System.

Chitin and chitosan have unique structures with significant functional groups carrying useful chemical capabilities. Chitin and chitosan are acknowledged as novel biomaterials with advantageous biocompatibility and biodegradability. Chitosan is a polysaccharide that is made from chitin. There have been several attempts to employ this biopolymer in the biomedical area. This material's application in the production of artificial skin, drug targeting, and other areas is explored. The most prevalent strategies for recovering chitin from sea organisms are described and various pharmacological and biological uses are discussed. This review article targets drug delivery with the help of chitosan derived nanomaterial. The drug delivery system applications through nonmaterial have encountered a considerable role in the pharmaceutical, medical, biological, and other sectors in recent years. Nanomaterials have advanced applications as novel drug delivery systems in many fields, especially in industry, biology, and medicine. In the biomedical and pharmaceutical arena, the natural polymer-based nanoparticulate method has now been widely studied as particulate vehicles. By mixing alginate with other biopolymers, by immobilizing specific molecules such as sugar molecules and peptides by chemical or physical cross-linking, different properties and structures such as biodegradability, gelling properties, mechanical strength, and cell affinity can be obtained. Owing to their inherent ability to deliver both hydrophilic and hydrophobic drug molecules, increase stability, decrease toxicity, and enhance commonly formulated medications, these particles are now widely used in imaging and molecular diagnostics, cosmetics, household chemicals, sunscreens, radiation safety, and novel drug delivery.

A Redox-Probe-Free Immunosensor Based on Electrocatalytic Prussian Blue Nanostructured Film One-Step-Prepared for Zika Virus Diagnosis.

The Zika virus (ZIKV) is a great concern for global health due to its high transmission, including disseminating through blood, saliva, urine, semen and vertical transmission. In some cases, ZIKV has been associated with microcephaly, neurological disorders, and Guillain−Barré syndrome. There is no vaccine, and controlling the disease is a challenge, especially with the co-circulation of the Dengue virus, which causes a severe cross-reaction due to the similarity between the two arboviruses. Considering that electrochemical immunosensors are well-established, sensitive, and practical tools for diagnosis, in this study we developed a sensor platform with intrinsic redox activity that facilitates measurement readouts. Prussian blue (PB) has a great ability to form electrocatalytic surfaces, dispensing redox probe solutions in voltammetric measurements. Herein, PB was incorporated into a chitosan−carbon nanotube hybrid, forming a nanocomposite that was drop-casted on a screen-printed electrode (SPE). The immunosensor detected the envelope protein of ZIKV in a linear range of 0.25 to 1.75 µg/mL (n = 8, p < 0.01), with a 0.20 µg/mL limit of detection. The developed immunosensor represents a new method for electrochemical measurements without additional redox probe solutions, and it is feasible for application in point-of-care diagnosis.

Assessment of avidity related to IgG subclasses in SARS-CoV-2 Brazilian infected patients.

SARS-CoV-2 is considered a global emergency, resulting in an exacerbated crisis in the health public in the world. Although there are advances in vaccine development, it is still limited for many countries. On the other hand, an immunological response that mediates protective immunity or indicates that predict disease outcome in SARS-CoV-2 infection remains undefined. This work aimed to assess the antibody levels, avidity, and subclasses of IgG to RBD protein, in symptomatic patients with severe and mild forms of COVID-19 in Brazil using an adapted in-house RBD-IgG ELISA. The RBD IgG-ELISA showed 100% of specificity and 94.3% of sensibility on detecting antibodies in the sera of hospitalized patients. Patients who presented severe COVID-19 had higher anti-RBD IgG levels compared to patients with mild disease. Additionally, most patients analyzed displayed low antibody avidity, with 64.4% of the samples of patients who recovered from the disease and 84.6% of those who died in this avidity range. Our data also reveals an increase of IgG1 and IgG3 levels since the 8th day after symptoms onset, while IgG4 levels maintained less detectable during the study period. Surprisingly, patients who died during 8-14 and 15-21 days also showed higher anti-RBD IgG4 levels in comparison with the recovered (P < 0.05), suggesting that some life-threatening patients can elicit IgG4 to RBD antibody response in the first weeks of symptoms onset. Our findings constitute the effort to clarify IgG antibodies' kinetics, avidity, and subclasses against SARS-CoV-2 RBD in symptomatic patients with COVID-19 in Brazil, highlighting the importance of IgG antibody avidity in association with IgG4 detection as tool laboratory in the follow-up of hospitalized patients with more significant potential for life-threatening.

Assessment of avidity related to IgG subclasses in SARS­CoV­2 Brazilian infected patients

SARS-CoV-2 is considered a global emergency, resulting in an exacerbated crisis in the health public in the world. Although there are advances in vaccine development, it is still limited for many countries. On the other hand, an immunological response that mediates protective immunity or indicates that predict disease outcome in SARS-CoV-2 infection remains undefned. This work aimed to assess the antibody levels, avidity, and subclasses of IgG to RBD protein, in symptomatic patients with severe and mild forms of COVID-19 in Brazil using an adapted in-house RBD-IgG ELISA. The RBD IgG-ELISA showed 100% of specifcity and 94.3% of sensibility on detecting antibodies in the sera of hospitalized patients. Patients who presented severe COVID-19 had higher anti-RBD IgG levels compared to patients with mild disease. Additionally, most patients analyzed displayed low antibody avidity, with 64.4% of the samples of patients who recovered from the disease and 84.6% of those who died in this avidity range. Our data also reveals an increase of IgG1 and IgG3 levels since the 8th day after symptoms onset, while IgG4 levels maintained less detectable during the study period. Surprisingly, patients who died during 8­14 and 15­21 days also showed higher anti-RBD IgG4 levels in comparison with the recovered (P< 0.05), suggesting that some life-threatening patients can elicit IgG4 to RBD antibody response in the frst weeks of symptoms onset. Our fndings constitute the efort to clarify IgG antibodies' kinetics, avidity, and subclasses against SARS-CoV-2 RBD in symptomatic patients with COVID-19 in Brazil, highlighting the importance of IgG antibody avidity in association with IgG4 detection as tool laboratory in the follow-up of hospitalized patients with more signifcant potential for life-threatening. (AU)

Mapping protective epitopes in the tick and mosquito subolesin ortholog proteins.

The tick protective antigen, subolesin, is a structural and functional ortholog of insect akirins, an evolutionary conserved group of proteins that regulate gene expression thus affecting multiple cellular processes such as digestion, immune response, reproduction and development. In this study, we searched for common protective epitopes in tick and mosquito subolesin ortolog proteins. By combining the results of peptide and phage-display libraries scan analysis with sera from protected animals with computational modeling, three different epitope types (i) linear B-cell epitopes, (ii) conformational epitopes, and (iii) conformational discontinuous epitopes were identified. The determination of conserved protective epitopes in subolesin ortologs may lead to the development of a multi-target universal vaccine directed at the control of both arthropod infestations and reduction of vector capacity to transmit pathogens.

Recombinant peptides as new immunogens for the control of the bovine tick, Rhipicephalus (Boophilus) microplus.

A candidate vaccine that effectively controls Rhipicephalus microplus ticks in livestock is an attractive strategy in bovine management because of the expensive and labor-intensive treatment of animals with acaricides. However, the complex nature of ectoparasites has imposed restrictions on the development of such a vaccine, and its future efficacy is still under debate. Using Phage Display technology, we developed specific immunogens that mimic R. microplus tick antigens and were successfully validated by in vitro and in vivo assays. Nine peptides were used in the experimental vaccination of mice and cattle with different formulations. The peptides generated specific antibody response against R. microplus larval proteins in mouse immunization. A vaccine mixture of all nine selected phage clones was employed in cattle vaccination and the immune response induced specific changes in teleogine physiology, manifested as a hemorrhagic event in the gastrointestinal and reproductive tracts. A new means for vaccine development and the discovery of immunogens have been demonstrated, and our data may provide evidence that a putative vaccine for ectoparasite control is possible.

A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection.

Toxoplasma gondii surface is coated by closely related antigens that belong to SRS (SAG-1 related sequences) superfamily. Two tachyzoite-specific SRS antigens, SAG1 and SAG2, are immunodominant proteins that apparently modulate the virulence of infection by inducing the host immune response against tachyzoites during the acute phase. In this study, we described a conformationally insensitive monoclonal antibody (A4D12mAb) that recognizes a linear epitope shared by two isoforms of p22 that is expressed in the surface of T. gondii tachyzoites. By using phage display approach and production of recombinant proteins, we clearly demonstrated that the A4D12mAb recognizes an epitope within C-terminal region of SAG2A. This mAb reacts with both T. gondii genotypes (I and II) but not with a closely related parasite, Neospora caninum. Also, the pretreatment of tachyzoites with A4D12 mAb did not inhibit T. gondii infection, suggesting that the epitope herein mapped is not crucial for tachyzoite invasion. However, a panel of human T. gondii positive sera showed significant degree of inhibition of A4D12 mAb reactivity against T. gondii native antigens, indicating that both A4D12 mAb and human sera recognize an overlapping immunodominant epitope within C-terminal region of SAG2A. To our knowledge, this is the first evidence using bioselection by phage display that identifies a T. gondii linear epitope recognized by a mAb specific to SAG2A. In conclusion, the results here presented add a new piece of information concerning T. gondii SAG2A molecule, emphasizing two dissimilar biological roles of this molecule, particularly for A4D12 epitope, suggesting that these characteristics may be important for parasite survival, since it is part of parasite components able to induce a strong immune response enough to allow host survival and establish long-term chronic infection.

Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations.

The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations.