Evaluation of RANTES analogue expression in Nicotiana benthamiana and Lycopersicon esculentum and their topical microbicidal activity

Abstract

The HIV/AIDS pandemic has dramatically altered patterns of morbidity and mortality in sub-Saharan Africa during the last two decades. In the absence of HIV vaccine, microbicides may offer viable option for protection against HIV infection. Microbicides are products that are applied topically inside the vagina or rectum that act to impede transmission of HIV and other sexually transmitted diseases. Small human chemokines such as RANTES (regulated upon activation, normal T cell expressed and secreted) are currently been investigated as microbicides candidates. A number of N-terminally modified RANTES analogues such as 5P12 and 6P4 with a much higher antiviral potency have been developed and they have strong potential for use as microbicides. Since plants offer an alternative option for cost effective production of protein therapeutics, we evaluated the feasibility of expressing 5P12 and 6P4 in Nicotiana benthamiana species. 5P12 is considered the most promising candidate for use in the microbicide pipeline because it inhibits HIV infection through cellular receptor antagonism. Hence its feasibility of expression was also evaluated in Lycopersicon esculentum (tomato). The two analogues were transiently expressed in the selected plant species via agrobacterium-mediated transfection. For expression in N. benthamiana, two different vectors (pTRA and MagnICON) were used to deliver the two analogues for transient expression. About 6-8 weeks-old N. benthamiana plants were agroinfiltrated via needle injection and vacuum infiltration methods and targeted to four subcellular compartments viz: apoplast, chloroplast, cytosol and endoplasmic reticulum (ER). The agroinfiltrated leaves were replanted, grown in a tissue culture laboratory and harvested after different periods. For expression in L. esculentum, the MagnICON constructs were used to deliver the 5P12 gene into four different developmental stages of tomato fruits viz: mature green (MG), breaker (B), pink (P) and ripe (R) via needle injection. The agroinjected tomato fruits were incubated in a dark cupboard and harvested after different periods. xiii Proteins were extracted from the harvested material and evaluated for 5P12 and 6P4 expression. ELISA results showed expression of 5P12 and 6P4 in N. benthamiana leaves which was detectable at 3-9 days post infiltration (dpi). Similar results were obtained for 5P12 and 6P4, consequently only results for 5P12 are reported. The vacuum infiltrated leaves of both pTRA and MagnICON constructs led to higher yields than the needle injected leaves. The highest yields were obtained with the MagnICON constructs. The highest 5P12 expression level of 603 μg/kg fresh weight leaf tissues (~0.024% TSP) was obtained in the apoplast at 9 dpi. The pTRA constructs had the highest expression levels of 0.63μg/kg FW in the cytosol at 3 dpi. 5P12 was also detectable at 3-9 dpi in L. esculentum, based on ELISA results. The highest 5P12 expression of 23.56 μg/kg FW and pH 4.75 tissues was obtained at the MG stage in the apoplast at 9 dpi. Western blot analysis confirmed the size of plantmade 5P12. Moreover, the plant extracts had anti-viral activity and were not toxic to TZM-bl cells. Our results show that the RANTES can be made in both N. benthamiana and L. esculentum and that the levels are not different from other systems reported previously. Furthermore, this is the first report that a chemokine has been expressed in plants. The quantities expressed were low making the commercial development of a microbicide from these species impractical. However, production of bulky leaf material may enhance the quantities.