Structure-activity, biochemical and antiviral studies on griffithsin to elucidate its mechanism of action and its specific binding parameters to oligosaccharides present on viral envelope glycoproteins. These structural determinations, done in collaboration with Dr. Alex Wlodawer (MCL), have allowed us to modify the GRFT structure to create mutants that have demonstrated varying degrees of anti-HIV activity and helped define the GRFT mechanism of action. Taken together, these results indicate that GRFT likely exhibits its potent antiviral activity by cross-linking oligosaccharides on the HIV-I envelope glycoprotein gp120 and preventing the subsequent conformational changes necessary for viral entry. The loss of activity of the monomer may be due to its inability to cross-link as efficiently as the native obligate GRFT dimer. This novel mechanism, revealed by our biophysical and structural studies, is supported by recent literature concerning GRFT induced mutations in gp120 oligosaccharide attachment sites and the dependence of lectin avidity on multivalent interactions. Studies towards the pre-clinical and clinical development of griffithsin as a topical anti-HIV microbicide. Since the large-scale production of GRFT, we have pursued its pre-clinical development through collaborations largely funded by NIAID. We have reported on its stability and efficacy in the mucosal environment, its activity in human cervical explant models and its safety and lack of either immune activation or epithelial irritation in multiple in vitro and in vivo model systems. This has resulted in several publications by other researchers detailing GRFTs surprising resistance to proteolytic degradation, its synergistic activity in combination with ARVs such as tenofovir and other lectins, and its enhancement of immune response to HIV-1 gp120. GRFT is now considered a leading candidate for human clinical trials. The formulation of GRFT as both a gel and a film, its stability and release from those formulations, its activity against a broad spectrum of strains of HIV, and its in vivo activity against HSV have also elevated its status. Currently, we are continuing the clinical development of GRFT as an anti-HIV microbicide. Evaluation of griffithsin for activity against HIV & HCV.-Previously we and others had shown that the antiviral lectin cyanovirin-N had activity against other enveloped viruses in addition to HIV. As GRFT was more potent than CV-N and, as a monosaccharide-specific lectin more promiscuous in its carbohydrate binding motif we proceeded to collaborate with NIAID to determine the in vitro antiviral spectrum of activity for GRFT. Simultaneously, we began a collaboration with Dr. Lynn Morris at NICD in S. Africa to test GRFT against circulating strains of HIV. The evaluation of GRFTs activity across the various clades of HIV-1 resulted in several interesting findings. Initially, we showed that, as with laboratory strains, clinical strains of HIV-1 in Clade C were very susceptible to GRFT at low nM to pM levels. Furthermore, virus in cervical vaginal lavages from HIV positive patients was also shown to be susceptible to inhibition by GRFT. Finally, our work evaluated the ability of GRFT (and our other lectins) to inhibit HIV-1 binding to the dendritic cell-associated lectin DC-SIGN. DC-SIGN is known to be a positive effector of HIV infection via the transfer and presentation of infectious virus to CD4+ T-cells. In this study we determined that GRFT was able to inhibit HIV-1 binding to DC-SIGN and to inhibit subsequent transfer of virus to CD4+ cells. The culmination of these experiments was a better understanding of both the scope and mechanism of GRFT activity against HIV and its potential utility as a anti-HIV microbicide. GRFT and another MTL discovery, SVN, were tested for activity against hepatitis C virus (HCV). This was a collaboration initiated with Dr. Yutaka Takebe at the National Institute of Infectious Disease in Japan. When tested in the HCV cell culture assay system, both SVN and GRFT were potently active with GRFT displaying an EC50= 50 pM and little toxicity. I received funding from NIAID to support in vivo testing of GRFT against HCV in a Alb-uPA-SCID mouse model with human hepatocyte engraftment. Injections of 20 mg/kg/day given to study animals for 10 days revealed that GRFT was non-toxic and rapidly bioavalable following s.c injection. We then completed two efficacy studies in which animals were dosed for either 10 or 18 consecutive days with GRFT. The results showed that 10 day treatment with GRFT, post HCV challenge, reduced HCV titers 100 fold. The 18-day GRFT treatment again reduced viral load by >100-fold, but resulted in significant toxicity to test animals (6/14 animals died) though not to the human liver tissue therein. Evaluation of the antiviral proteins griffithsin and scytovirin for activity against ebola.- I have an ongoing collaboration with both USAMRIID and NIAID to investigate the proteins we have discovered for activity against pathogenic viruses of potential biodefense interest. Part of this work has been funded by a Trans-NIH grant that I received from NIAID to work on the use of the proteins SVN and GRFT against the ebola virus. Ebola virus is a category A infectious agent with no approved therapeutic options and has a mortality rate in humans of >50%. Initial in vitro pseudoparticle assays on SVN and GRFT against ebola Zaire and Marburg virus showed that these proteins had nanomolar activity against both filoviruses. Tolerability studies in mice indicated that GRFT and SVN were well tolerated at doses up to 40 mg/kg/day with GRFT being dosed either Q12 or Q24 and SVN dosed Q6. Efficacy studies in mice with both proteins in various dose ranges indicated that treatment with either SVN or GRFT for 10 days resulted in survival rates of 90% for treated animals (compared to 0% for control animals). Isolation, characterization and cloning of anti-HIV proteins isolated from natural products extracts. The aqueous extract of Synthecium sp. showed anti-HIV activity and yielded 3 novel anti-HIV proteins. The purified cnidarin proteins were named cnidarin 1-3 (CNID-1, CNID-2, CNID-3). The proteins, homogenous by SDS-PAGE, showed single peaks for each protein by ESI/MS, corresponding to exact molecular masses of 18,122 Da (CNID-1), 18,088 Da (CNID-2) and 17,963 Da (CNID-3). Amino acid sequences of the purified CNID proteins were established. The peptide fragments for CNID-1 and CNID-3 sufficiently overlapped to sequence both proteins. CNID-1 and CNID-3 share a 71% sequence similarity when aligned against each other, and when compared against known proteins using the NCBI database they showed no significant sequence homology to any known protein. All three CNID proteins elicited concentration-dependent inhibition of virus-induced cell killing with picomolar EC50 values. The CNIDs were remarkably potent with picomolar to low-nanomolar range activity against HIV, which is, on average, lower or comparable to the activity of other antiviral proteins isolated from natural sources, with the exception of griffithsin. Of the three CNID proteins, CNID-1 was the most potent at protecting against HIV-1RF-induced cytopathic effects in CEM-SS cells (EC50 = 85 pM). CNID-3 was most potent at inhibiting the HIV-1ROJO primary isolate (EC50 of 1.5 nM) while CNID-2 was the least potent. CNID-1 bound to gp41 and gp120 equivalently well which is distinct from our previous discoveries of CV-N, SVN and GRFT. I am also on the steering committee of two clinical trials that have been funded for the evaluation of griffithsin as an anti-HIV microbicide. Additional intramural studies on GRFT activity against Nipah virus, ebola and MERS have been initiated. We have also collaborted extramurally on activity against HSV-1 & 2 and Trichnomas vaginalis.
