Studies in a Murine Model Confirm the Safety of Griffithsin and Advocate Its Further Development as a Microbicide Targeting HIV-1 and Other Enveloped Viruses

Kouokam, Joseph Calvin; Lasnik, Amanda B.; Palmer, Kenneth E.

All mice survived single treatments with up to 50 mg/kg, and 14 daily treatments of 10 mg/kg GRFT. In addition, no GRFT treatment related adverse effects in behavior or fitness (body-weight gain or loss) were observed. These findings suggest that GRFT does not cause massive toxicity in mice after parenteral administration. Of note, the amounts of proteins used in these experiments were very high, since we aimed at recording the maximal effects that the lectin could trigger in adult mice. Recently, we demonstrated that GRFT is tolerated with minimal toxicity in juvenile guinea pigs after subcutaneous administration of 10 daily doses of 10 mg/kg [17] . Other lectins have also been studied in mice for their effects after parenteral administration. For instance, in an attempt to determine a well-tolerated dose in mice, Cyanovirin-N (CV-N) was injected beneath the skin of the upper back, and daily doses of 14 mg/kg caused weight loss in mice, while doses of 4.8 mg/kg and lower did not cause any signs of illness [20] . Our data revealed no GRFT treatment-related toxic effects on lungs, livers, kidneys, and hearts, as evaluated by organ weights and/or histology. However reversible splenomegaly was observed in mice treated with a single high dose (50 mg/kg) or chronically with 10 mg/kg GRFT. A study involving Phaseolus vulgaris lectin revealed that parenteral administration of the carbohydrate binding protein affects the extra-intestinal organs in suckling rats [21] . Indeed, it was shown that liver and spleen weights increased in animals treated parenterally with phytohemagglutinin and the authors suggested that the changes in organ weights are the result of an immunological cleansing/response process of these organs in response to the circulating PHA after the s.c. administration [21] . It is plausible that the observed splenomegaly in the case of GRFT reflects a nascent immune reaction from the animals as well. As shown above, B220-only positive splenocytes (CD19-) were elevated after 14 daily treatments with GRFT at 10 mg/kg, a difference that was not obtained after a week of recovery. Recently, a subset of murine splenic cells expressing B220 and lacking CD19 was described by Murakami and collaborators, and proposed to be involved in the formation of a follicular dendritic cell network [22] , which plays a critical role in the immune reaction to foreign antigens [23] . It is unclear whether CD19 −/ B220 + cells increased after GRFT treatment have the same function in our model. In addition, CD69 expressing cells were slightly more abundant after chronic treatment with 10 mg/kg GRFT in this study; this effect was reduced after the recovery period. The effect of CD69 on spleen size remains controversial. While it was demonstrated that induction of the T-cell early activation marker CD69 in mice results in marked splenomegaly [24] , CD69 knockout mouse spleens were shown to be enlarged compared with those of WT mice in tumor and collagen-induced arthritis challenge models [25, 26] .