A DNA copy number alteration classifier as a prognostic tool for prostate cancer patients.

BACKGROUND: Distinguishing between true indolent and potentially life-threatening prostate cancer is challenging in tumours displaying clinicopathologic features associated with low or intermediate risk of relapse. Several somatic DNA copy number alterations (CNAs) have been identified as potential prognostic biomarkers, but the standard cytogenetic method to assess them has a limited multiplexing capability. METHODS: Multiplex ligation-dependent probe amplification (MLPA) targeting 14 genes was optimised to survey 448 tumours of patients with low or intermediate risk (Grade Group 1-3, Gleason score ≤7) who underwent radical prostatectomy. A 6-gene CNA classifier was developed using random survival forest and Cox proportional hazard modelling to predict biochemical recurrence. RESULTS: The classifier score was significantly associated with biochemical recurrence after adjusting for standard clinicopathologic variables and the known prognostic index CAPRA-S score with a hazard ratio of 2.17 and 1.80, respectively (n = 406, P < 0.01). The prognostic value of this classifier was externally validated in published CNA data from three radical prostatectomy cohorts and one radiation therapy pre-treatment biopsy cohort. CONCLUSION: The 6-gene CNA classifier generated by a single MLPA assay compatible with the small quantities of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens has the potential to improve the clinical management of patients with low or intermediate risk disease.

Design, expression and evaluation of novel chimeric protein constructed from colorectal tumor-associated antigen.

There were 134,000 new diagnosis and 49,000 deaths in 2016 due to colorectal cancer. Similar to most cancers, early diagnosis increases the chance of successful treatment. Detection of tumor-associated antigens or the immune response against such markers is one of the most common methods of diagnosis. In that regard, we aimed to design and express a chimeric protein from the most common tumor-associated antigens in colorectal cancer and assess its ability to detect the immune response in comparison with the parental tumor-associated antigens in patient's sera. Through bioinformatics approaches a chimeric protein from carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA19-9) was designed and expressed in E. coli (BL21DE3). Proper folding, expression levels and immune reactivity were assessed by western blot, ELISA and immunohistochemistry. Recombinant proteins functionality and immune reactivity were confirmed by ELISA and Western blot. Results showed that recombinant CEA, recombinant CA19.9 and chimeric protein of CEA- CA19.9 have strong reactivity with antibodies in the sera of colorectal cancer patients, whereas no reactivity was seen with the sera of healthy volunteers. Significantly stronger immune reactivity was seen with the chimeric protein than each of the CEA or CA19.9 alone. Overall, it was concluded that the designed recombinant proteins in this study could be used to detect autoantibodies produced against the colorectal tumor-associated antigens. The chimeric CEA-CA19.9 protein shows a stronger reactivity with the sera antibodies of colorectal cancer patients that CEA or CA19.9 alone.

Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10µg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

A conserved region from biofilm associated protein as a biomarker for detection of Acinetobacter baumannii.

Acinetobacter baumannii is the leading cause of nosocominal infection within the family Moraxellaceae. Due to multiple antibiotic resistances of the bacteria, the treatment is very difficult, hence specific and economical test for early diagnosis of infection is needed. Development of such a test requires targeting specific cell surface antigens. Bacterial ability of biofilm formation grants major contribution in antimicrobial resistance and other environmental stresses such as nutrient limitation and dehydration. Biofilm associated protein (Bap), a specific cell surface protein, is directly involved in A. baumannii biofilm formation. The goal of this study is diagnosis of A. baumannii infection exploiting specific target from Bap. A selected subunit of Bap was cloned, expressed and purified. Mice were divided into three groups. Group one was immunized with recombinant Bap subunit, mice in group two were infected with A. baumannii (positive control) and mice in groups three served as negative control. Immunization with Bap subunit resulted in high antibody titers. Animals in control group that received same amount of adjuvant and PBS showed no Bap-specific antibodies. Sensitivity and specificity of the antibodies raised were determined by ELISA and Western blotting. Recombinant Bap subunit was tested by ELISA using sera obtained from A. baumannii infected patients and healthy individuals. This conserved and immunodominant region of Bap could serve as an appropriate target for diagnosis A. baumannii infection.

Bacteriophage vehicles for phage display: biology, mechanism, and application.

The phage display technique is a powerful tool for selection of various biological agents. This technique allows construction of large libraries from the antibody repertoire of different hosts and provides a fast and high-throughput selection method. Specific antibodies can be isolated based on distinctive characteristics from a library consisting of millions of members. These features made phage display technology preferred method for antibody selection and engineering. There are several phage display methods available and each has its unique merits and application. Selection of appropriate display technique requires basic knowledge of available methods and their mechanism. In this review, we describe different phage display techniques, available bacteriophage vehicles, and their mechanism.

A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis.

Expression of carbonic anhydrase IX (CAIX) significantly increases under hypoxic conditions in tumor cells. CAIX activity is executed by the catalytic domain (CA) located on the extracellular part of the enzyme. Neutralization of CAIX enzymatic activity reduces malignancy and survival of tumor cells. To inhibit the enzymatic activity, a VHH nanobody was developed against the CA domain of CAIX using phage display technology. Following immunization of a camel with the recombinant CAIX, VHH fragments were isolated by nested PCR on lymphocyte cDNA. Binding affinity of isolated nanobodies was tested by ELISA. A clone (K24) with the highest binding affinity was expressed in a soluble form. Affinity of K24 nanobody was determined to be approx. 2.3 × 10(-5). K24 nanobody recognized the expressed CAIX in the HeLa cell lines with high selectivity and specificity. These findings thus have usefulness for the diagnosis and treatment of cancers.

Investigating the effect of cGRP78 vaccine against different cancer cells and its role in reducing melanoma metastasis.

Background and purpose: Treatment of malignancies with chemotherapy and surgery is often associated with disease recurrence and metastasis. Immunotherapy improves cancer treatment by creating an active response against tumor antigens. Various cancer cells express a large amount of glucose-regulated protein 78 (GRP78) protein on their surface. Stimulating the immune system against this antigen can expose cancer cells to the immune system. Herein, we investigated the effectiveness of a cGRP78-based vaccine against different cancer cells. Experimental approach: BALB/c mice were immunized with the cGRP78. The humoral immune response against different cancer cells was assessed by Cell-ELISA. The cellular immunity response was determined by splenocyte proliferation assay with different cancer antigens. The effect of vaccination on metastasis was investigated in vaccinated mice by injecting melanoma cancer cells into the tail of mice. Findings/Results: These results indicated that the cGRP78 has acceptable antigenicity and stimulates the immune system to produce antibodies. After three injections, the amount of produced antibody was significantly different from the control group. Compared to the other three cell types, Hela and HepG2 showed the highest reaction to the serum of vaccinated mice. Cellular immunity against the B16F10 cell line had the best results compared to other cells. The metastasis results showed that after 30 days, the growth of B16F10 melanoma cancer cells was not noticeable in the lung tissue of vaccinated mice. Conclusion and implications: Considering the resistance of vaccinated mice to metastasis, this vaccine offers a promising prospect for cancer treatment by inhibiting the spread of cancer cells.

Introduction of a new recombinant vaccine based on GRP78 for breast cancer immunotherapy and evaluation in a mouse model.

Introduction: Breast cancer is one of the most prevalent malignancies in women. Several treatment options are available today, including surgery, chemotherapy, and radiotherapy. Immunotherapy, as a highly specific therapy, involves adaptive immune responses and immunological memory. In our present research, we used the recombinant C-terminal domain of the GRP78 (glucose- regulated protein 78) protein to induce an immune response and investigate its therapeutic impact in the 4T1 breast cancer model. Methods: BALB/c mice were immunized with the cGRP78 protein. The humoral immune response was assessed by ELISA. Then, BALB/c mice were injected subcutaneously with 1×106 4T1 tumor cells. Subsequently, tumor size and survival rate measurements, MTT, and cytokine assays were performed. Results: The animals receiving the cGRP78 vaccine showed significantly more favorable survival and slower tumor growth rates compared with unvaccinated tumor-bearing mice as the negative control mice. Circulating levels of tumoricidal cytokines such as IFNγ were higher, whereas tolerogenic cytokines such as IL-2, 6, and 10 either did not increase or had a decreasing trend in mice receiving cGRP78. Conclusion: cGRP78 vaccines generated potent immunotherapeutic effects in a breast cancer mouse model. This novel strategy of targeting the GRP78 protein can promote the development of cancer vaccines and immunotherapies for breast cancer malignancies.

Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes.

Vibrio cholerae is considered one of the major health threats in developing countries. Lack of efficient vaccine, short incubating time of the disease, and bacterium ability to survive in aquatic environment have made cholera one of the most epidemic diseases yet known. The lipopolysaccharide is one of the bacterium key antigens used to classify V. cholerae into 206 serogroups. V. cholerae serogroup O1 is a causative agent of all cholera pandemics. Research has shown that anti-lipopolysaccharide (LPS) antibodies could provide protective immunity in cholera cases. In this research, we used N-terminal fragments of the camel's heavy-chain antibodies called VHH or nanobodies and produced a phagemid library. The obtained library was panned against V. cholerae O1 LPS, and four monoclonal nanobodies were isolated. Isolated nanobodies were tested in LPS ELISA and bacterial ELISA. The nanobody with the highest affinity toward the bacterium was used in an in vivo challenge and successfully neutralized the bacterium infection. The isolated nanobody showed high thermostability and proteolytic resistance in characterization tests.

Maveropepimut-S, a DPX-Based Immune-Educating Therapy, Shows Promising and Durable Clinical Benefit in Patients with Recurrent Ovarian Cancer, a Phase II Trial.

PURPOSE: Patients with platinum-resistant ovarian cancer respond poorly to existing therapies. Hence there is a need for more effective treatments. PATIENTS AND METHODS: The DeCidE1 trial is a multicenter, randomized, open-label, single-arm phase II study to evaluate the safety and effectiveness of maveropepimut-S with cyclophosphamide in patients with recurrent ovarian cancer. Median follow-up for evaluable subjects was 4.4 months. Data were collected from March 2019 to June 2021. Subjects received two injections of 0.25 mL maveropepimut-S 3 weeks apart, followed by one 0.1-mL doses, every 8 weeks up to progression. Oral cyclophosphamide, 50 mg twice daily, was administered in repeating weekly on and off cycles. RESULTS: Twenty-two patients were enrolled. Median age was 58 years (38-78 years). Among the evaluable population, the objective response rate (ORR) was 21% [90% confidence interval (CI), 7.5%-41.9%], with a disease control rate (DCR) of 63% (90% CI, 41.8%-81.3%), including 4 (21%) patients with partial responses, 8 (42%) stable disease, and 7 (37%) progressive disease. The ORRs were consistent across subgroups based on platinum sensitivity, and DCR was higher in the platinum-resistant subpopulation. Four SD patients maintained clinical benefit up to 25 months. Most treatment-related adverse events (TRAE) were grade 1 and 2 (87% of unique events). Most common AEs were injection site reactions. Eight subjects reported grade 3 and no grade 4 AEs. Survivin-specific T-cell responses were observed in treated patients with clinical benefit. CONCLUSIONS: Maveropepimut-S with intermittent low-dose cyclophosphamide is well-tolerated, with clinical benefit for patients with recurrent ovarian cancer. Observed responses are irrespective of the platinum status.

Inhibition of H. pylori colonization and prevention of gastritis in murine model.

Helicobacter pylori is a Gram-negative spiral bacterium that colonizes human gastric mucosa causing infection. In this study aiming at inhibition of H. pylori infection we made an attempt to evaluate immunogenicity of the total (UreC) and C-terminal (UreCc) fragments of H. pylori urease. Total UreC and its C-terminal fragment were expressed in E. coli. Recombinant proteins were analyzed by SDS-PAGE and western blot and then purified by Ni-NTA affinity chromatography. Female C57BL6/j mice were immunized with the purified proteins (UreC and UreCc). Antibody titers from isolated sera were measured by ELISA. Immunized mice were then challenged by oral gavage with live H. pylori Sydney strain SS1. Total of 109 CFU were inoculated into stomach of immunized and unimmunized healthy mice three times each at one day interval. Eight weeks after the last inoculation, the blood sample was collected and the serum antibody titer was estimated by ELISA. Stomach tissues from control and experimental animal groups were studied histopathologically. UreC and UreCc yielded recombinant proteins of 61 and 31 kDa respectively. ELIZA confirmed establishment of immunity and the antibodies produced thereby efficiently recognized H. pylori and inhibited its colonization in vivo. Pathological analysis did not reveal established infection in immunized mice challenged with H. pylori. The results support the idea that UreC and UreCc specific antibodies contribute to protection against H. pylori infections.

Treatment of Helicobacter pylori infection in mice with oral administration of egg yolk-driven anti-UreC immunoglobulin.

BACKGROUND: Helicobacter pylori, the causative agent of gastritis and gastric ulcer, plays a crucial role in development of gastric carcinomas. Antibiotic therapy fails in almost 20% of cases due to development of antibiotic resistance. Development of antibodies against specific H. pylori targets could have significant therapeutic effect. In the present research attempts have been made to study the effect of IgY purified from egg yolk of hens immunized with recombinant UreC in treatment of mice infected with H. pylori. MATERIALS AND METHODS: Purified IgY-HpUc was used in two forms: powdered and PBS dissolved. 10(9) bacteria in BHI were orally administered to C57BL6/j mice three times on alternate day intervals. Eight weeks after the last inoculation, the serum was assayed for infection rate by ELISA. The severity of gastritis was analyzed histopathologically. Infected mice were randomly divided into three groups. Groups one and two were treated with dietary IgY-HpUc and IgY-HpUc dissolved in PBS respectively for 28 days. The untreated group served as control. RESULTS: Serology and histopathology confirmed the establishment of the infection. Indirect ELISA results in the treated animals showed considerable reduction of H. pylori specific antibodies in their sera. Pathological examination of gastric mucosa of infected mice treated with IgY-HpUc showed considerable reduction of inflammation in the stomach tissues. The bacterial presence on mucosal layer of the stomach was considerably reduced. CONCLUSIONS: UreC-induced IgY is specifically successful in inhibition of H. pylori infection and could be an alternative to antibiotic treatment.

Expression of recombinant G-CSF receptor domains and their inhibitory role on G-CSF function.

BACKGROUND AND PURPOSE: Granulocyte colony-stimulating factor (G-CSF) is routinely used in combination with chemotherapy to battle neutropenia. However, studies suggest that this chemokine may increase the risk of metastasis and malignancy in many cancers. To counteract the adverse effects of G-CSF in cancer, antibodies have been used to block its action. However, antibodies are large and complex molecules which makes their production expensive. Thus in this study, we aim to construct different structure variants of the G-CSF receptor containing different domains and select the best variant that prevents the adverse actions of this chemokine. These novel structures are smaller than antibodies and easier to produce. EXPERIMENTAL APPROACH: Different domains of the G-CSF receptor were designed and cloned into the pET28a expression vector. These recombinant receptor subunits were then expressed in Escherichia coli and purified using standard affinity chromatography techniques. Interaction of recombinant receptor subunits with G-CSF was assessed using enzyme-linked immunosorbent assay and NFS60 cells. FINDINGS / RESULTS: Two recombinant receptor subunits containing D1 + D2 + D3 domains and D2 domain showed the strongest inhibitory activity to G-CSF. CONCLUSION AND IMPLICATIONS: These novel recombinant receptor variants could be candidates for further studies in the development of novel therapeutics.

Design and Development of a Fully Synthetic Multiplex Ligation-Dependent Probe Amplification-Based Probe Mix for Detection of Copy Number Alterations in Prostate Cancer Formalin-Fixed, Paraffin-Embedded Tissue Samples.

DNA copy number alterations (CNAs) are promising biomarkers to predict prostate cancer (PCa) outcome. However, fluorescence in situ hybridization (FISH) cannot assess complex CNA signatures because of low multiplexing capabilities. Multiplex ligation-dependent probe amplification (MLPA) can detect multiple CNAs in a single PCR assay, but PCa-specific probe mixes available commercially are lacking. Synthetic MLPA probes were designed to target 10 CNAs relevant to PCa: 5q15-21.1 (CHD1), 6q15 (MAP3K7), 8p21.2 (NKX3-1), 8q24.21 (MYC), 10q23.31 (PTEN), 12p13.1 (CDKN1B), 13q14.2 (RB1), 16p13.3 (PDPK1), 16q23.1 (GABARAPL2), and 17p13.1 (TP53), with 9 control probes. In cell lines, CNAs were detected when the cancer genome was as low as 30%. Compared with FISH in radical prostatectomy formalin-fixed, paraffin-embedded samples (n = 18: 15 cancers and 3 matched benign), the MLPA assay showed median sensitivity and specificity of 80% and 93%, respectively, across all CNAs assessed. In the validation set (n = 40: 20 tumors sampled in two areas), the respective sensitivity and specificity of MLPA compared advantageously with FISH and TaqMan droplet digital PCR (ddPCR) when assessing PTEN deletion (FISH: 85% and 100%; ddPCR: 100% and 83%) and PDPK1 gain (FISH: 100% and 92%; ddPCR: 93% and 100%). This new PCa probe mix accurately identifies CNAs by MLPA across multiple genes using low quality and quantities (50 ng) of DNA extracted from clinical formalin-fixed, paraffin-embedded samples.

Production of human single-chain fragment antibody (ScFv) against human epidermal growth factor receptor-2 (HER-2) by phage display technology.

Breast cancer with more than 1.7 million diagnoses per year has been known as one of the most prevalent cancers among women worldwide. Despite the availability of advanced treatment options, cancer progression and metastasis is observed in 20% of patients. Human epidermal growth factor receptor-2 (HER-2) is considered as an important prognostic and diagnostic tumor marker for breast cancer. While HER-2 is expressed on the surface of normal cells, its overexpression occurs in 20-25% on breast cancer tumor cells. This type of tumor which is referred to as HER-2+ is the most aggressive type of breast cancer and shows more resistance to radiotherapy. Single-chain fragment antibodies (ScFvs) offer several advantages in comparison to conventional whole antibodies due to their small size. Particularly, ScFv fragments show improved diffusion and solid tumor penetration. In this study, a human ScFv antibody library was used to isolate anti-HER-2 ScFv antibodies through cell panning and mix antigen-cell panning strategies. Analysis of the binding activity and specificity of isolated ScFv antibodies against HER-2-expressing cell lines and recombinant HER-2 antigen indicated the higher efficiency of the cell panning strategy in isolation of ScFv antibody fragments.

Targeting Colorectal Cancer Cell Lines Using Nanobodies; AgSK1as a Potential Target.

Background: Colorectal cancer is the third most common type of aggressive cancers. Chemotherapy, surgery, and radiotherapy are the common therapeutic options for treating this cancer. Due to the adverse side-effects of these methods, immunotherapy is considered as an appropriate alternative therapeutic option. Treatment through the application of monoclonal antibodies is considered as a novel alternative therapeutic method for cancers. The variable fragments of the antibodies' heavy chain or VHHs have a wide application in molecular biology and biotechnology. VHHs are compatible with the phage display technology which allows rapid and high throughput screening for antibodies isolation. Objectives: We aimed to use naive VHH phage library to isolate a specific nanobody against colorectal tumor associated antigen; the AgSK1. Materials and Methods: In this research, naive VHH phage library was panned against two colorectal cell lines; Ls174T and HT29 expressing different levels of AgSK1 tumor associated marker. The high affinity binders were selected and subcloned for higher expression levels of the VHH. The affinity and specificity of the isolated VHH were tested using ELISA. The reactivity of the VHH toward cancer cells was analyzed by competitive ELISA applying sera isolated from colorectal cancer patients. Results: Results show that the isolated VHH recognizes and binds to the colorectal cancer cells with a high affinity. Moreover, the isolated nanobody is able to compete with the antibodies in the patient sera for the binding to the cancer cells. Conclusions: Results suggest that this nanobody has a specific reaction toward colorectal cells and can be used for further investigation on the tumor associated antigens or production of mimotopes useful for immunotherapy.

Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models.

Selection of Single Chain Antibody Fragments for Targeting Prostate Specific Membrane Antigen: A Comparison Between Cell-based and Antigen-based Approach.

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second most common cause of cancer related mortality in United States male population. ScFv fragments have different usefulness. For example they have small size, high perfusion rate, high yield of production and are non-immunogenic, thus they can be used for therapeutic purposes. In this project we used a synthetic human ScFv library for isolation of ScFv monoclonal antibodies against prostate specific membrane antigen. For this purpose, after five rounds of cell-panning, and also five rounds of antigen-panning with rPSMA specific anti- PSMA ScFv-phage particles were isolated. Phages with high affinity toward PSMA were selected and used for further analysis. Specificity and affinity of both ScFv to PSMA and LnCaP cell line examined by ELISA. Recombinant ScFv antibody isolated from cell-panning had higher specificity and affinity for both the antigen and LNCaP cell line. Our result demonstrated that ScFv antibody obtained by cell-panning can target PSMA antigen and cell lines.

Production of Novel VHH Nanobody Inhibiting Angiogenesis by Targeting Binding Site of VEGF.

Vascular endothelial growth factor (VEGF) is one of the key players in angiogenesis and is considered as one of the major targets in cancer therapy. VEGF is secreted by the cancerous cells to form new vessels that carry oxygen and nutrients to the tumor, allowing it to grow beyond 1-2 mm. Cancerous cells spread using these veins and cause malignancy. Therefore, neutralization of VEGF could prevent tumor growth and malignancy, and nowadays, antibodies are widely used for such purpose. Among antibody fragments, nanobodies possess unique characteristics which make them appropriate tools for cancer therapy. In this study, the receptor-binding region of VEGF was used to produce a nanobody using phage display technology. A camel was immunized with the recombinant VEGF, and VHH fragments were amplified using nested PCR on lymphocyte complementary DNA (cDNA). The highest binding affinity was achieved after three rounds of panning. Twenty-four clones were tested by monoclonal phage ELISA, and the clone with the highest affinity (VA12) was selected for soluble expression of nanobody. VA12 was tested under various physicochemical conditions and showed considerable stability in extreme temperatures, pH, and various urea concentrations. Stability of VA12 under such conditions is considered as an advantage over the prevailing antibodies.

Immunogenicity of conserved cork and ß-barrel domains of baumannii acinetobactin utilization protein in an animal model.

BACKGROUND/AIM: The uptake of the ferric-acinetobactin complex into the periplasmic space relies on the baumannii acinetobactin utilization (BauA) protein. BauA is composed of cork and the ß-barrel domains. We constructed a recombinant protein from conserved antigenic domains of cork and the ß-barrel of BauA to evaluate their immunogenic role in an animal model. MATERIALS AND METHODS: The selected bauA domains were amplified from a purified genome of Acinetobacter baumannii ATCC 19606. The domains were then cloned into pET28a and the proteins expressed in E. coli BL21 (DE3) were purified using nickel nitrilotriacetic acid chromatography. Mice and rabbits were immunized with an intraperitoneal injection of the recombinant BauA (rBauA). RESULTS: The highest immune response was achieved after the third booster injection while hyperimmunity was achieved after the second booster injection in rabbits. Immunized mice challenged with live A. baumannii survived, whereas all unimmunized mice in the control group died after 24 h. Mice injected with 10(9) colony forming units of A. baumannii preincubated with pure immune rabbit sera survived. Bacterial cultures from mice spleen and liver specimens revealed the absence of bacterial growth in the immunized groups. CONCLUSION: The rBauA could be used as a prophylactic agent and further tests should be carried out to see if it may be useful in a clinical setting against A. baumannii infections.