Synthesis and characterization of a novel chemically designed (Globo)3–DTPA–KLH antigen

Mehdi Hajmohammadi,1 Seyed Davar Siadat,2 Masoud Ghorbani,3,4,* Mehdi Shafiee Ardestani,5,* Shahram Teimourian,6 Vahid Asgari,3 Reza Ahangari Cohan,3 Mostafa Hajmohammadi,5 Akram Hajmohammadi,7 Ramezan Behzadi,8 Saied Rajab Nezhad,9 Nabiollah Namvar Asl10 1Department of Research and Biotechnology, 2Department of Microbiology, 3Department of Virology, Pasteur Institute of Iran, Tehran Iran; 4Department of Virology, University of Ottawa, Ottawa, ON, Canada; 5Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 6Department of Medical Genetics, Iran University of Medical Sciences, Tehran, 7Faculty of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, 8Laboratory Animal Management of North Research Center, Pasteur Institute of Iran, 9Department of Research and Development, Alhavi Pharmaceutical, Tehran, 10Pasteur Institute of Iran, Department of Animal Sciences, Karaj, Iran *These authors contributed equally to this work Abstract: In recent years, many experiments have been conducted for the production and evaluation of anticancer glycoconjugated vaccines in developed countries and many achievements have been accomplished with Globo H derivatives. In the current experiment, a new chemically designed triplicate version of (Globo H)3–diethylenetriamine pentaacetic acid (DTPA)–KLH antigen was synthesized and characterized. Immunization with (Globo H)3-DTPA-KLH, a hexasaccharide that is a member of a family of antigenic carbohydrates that are highly expressed in various types of cancers conjugated with DTPA and KLH protein, induced a high level of antibody titer along with an elevated level of IL-4 in mice. Treatment of tumors with the collected sera from immunized mice decreased the tumor size in nude mice as well. None of the immunized mice illustrated any sign of tumor growth after injection of MCF-7 cells compared to the control animals. These findings, based on the newly presented structure of the Globo H antigen, lend exciting and promising evidence for clinical advancement in the development of a therapeutic vaccine in the future. Keywords: (Globo H)3-DTPA-KLH, glycoconjugate vaccines, breast cance

Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant <i>L. tarentolae</i> Vaccine against Experimental Murine Visceral Leishmaniasis

<div><p>Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans <i>Leishmania (L.) tarentolae</i> species as a live vaccine vector to deliver specific <i>Leishmania</i> antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against <i>L. infantum</i> infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant <i>L. tarentolae</i> expressing the <i>L. donovani</i> A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB^-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB^-CTE-recombinant <i>L. tarentolae</i> protects BALB/c mice against <i>L. infantum</i> challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB^-CTE-recombinant <i>L. tarentolae</i> as a safe live vaccine candidate against VL.</p> </div

Synthesis and characterization of a novel chemically designed (Globo)3–DTPA–KLH antigen

In recent years, many experiments have been conducted for the production and evaluation of anticancer glycoconjugated vaccines in developed countries and many achievements have been accomplished with Globo H derivatives. In the current experiment, a new chemically designed triplicate version of (Globo H)3–diethylenetriamine pentaacetic acid (DTPA)–KLH antigen was synthesized and characterized. Immunization with (Globo H)3-DTPA-KLH, a hexasaccharide that is a member of a family of antigenic carbohydrates that are highly expressed in various types of cancers conjugated with DTPA and KLH protein, induced a high level of antibody titer along with an elevated level of IL-4 in mice. Treatment of tumors with the collected sera from immunized mice decreased the tumor size in nude mice as well. None of the immunized mice illustrated any sign of tumor growth after injection of MCF-7 cells compared to the control animals. These findings, based on the newly presented structure of the Globo H antigen, lend exciting and promising evidence for clinical advancement in the development of a therapeutic vaccine in the future

Expression of the A2-CPA-CPB^-CTE-EGFP tri-fusion gene by <i>L.tarentolae</i>.

<p>(A) Expression of EGFP by recombinant <i>L. tarentolae</i>-EGFP promastigotes (left) and <i>L. tarentolae</i>-A2-CPA-CPB^-CTE-EGFP promastigotes (right) before and after glinting of fluorescence. (B) Percentage of the EGFP positive population in <i>L. tarentolae</i> transfected with either pLEXSY-EGFP (left) or pLEXSY-A2-CPA-CPB^-CTE-EGFP (clone #5, right) as determined by flow cytometry. (C) Western blot analysis for evaluating expression of the A2-CPA-CPB^-CTE-EGFP fusion protein. A 102.56 kDa band corresponding to the A2-CPA-CPB^-CTE-EGFP protein was detected in the recombinant <i>L. tar</i>-A2-CPA-CPB^-CTE-EGFP by western blotting using an anti-CPB antibody. No band was seen in lane 1 representing a negative control (<i>L. tarentolae</i> wild type).</p

Degree of inflammatory cell infiltration in liver parenchyma of all groups at 4 weeks after challenge.

<p>G1 to G5 groups are as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002174#pntd-0002174-g002" target="_blank">Figure 2</a>. The number of independent repeats was two and all tests were done in duplicate (number of mice per group/time point n = 2) and the results are pooled and shown as mean±S.E. of measures obtained from 4 mice of each group. The asterisk indicates the significant difference between values at the indicated time points as determined by Student's test (<i>p</i><0.05 denoted as *, <i>p</i><0.01 denoted as **, <i>p</i><0.001 denoted as *** and n.s. denoted as non significant).</p