Researchers at the University of Central Florida have used proteomic techniques to identify proteins commonly found in vaginal fluid that, when kept together as a group, play a role in protecting against HIV infection.
When the proteins are combined with a topical anti-HIV antimicrobial, there may be a synergistic anti-HIV effect, said Alexander Cole, the leader of the study who is an assistant professor in the department of molecular biology and microbiology at UCF.
"The hope is that [the topical antimicrobial] will work better than expected when applied to the vagina because of synergism with these proteins," said Cole. "Initial data indicate that there might be some synergy between some of our peptides and the [active ingredient] of the topical antimicrobial."
Cole, whose lab is developing the antimicrobial, has been studying a particular type of protein called cationic polypeptides for the past 11 years. Most peptides with antimicrobial activity tend to be cationic, he explained.
In his recent study, published in the Dec. 1 issue of the Journal of Immunology, Cole investigated the role that cationic polypeptides in vaginal fluid play in protecting against HIV-1. He and his colleagues first tested the anti-HIV-1 activity of vaginal fluid that was close to its natural form. They found that the fluid has intrinsic anti-HIV-1 properties it was capable of reducing the integration of the viral genome into host cell genomes in cultures of human cervicovaginal tissue.
"These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluids."
The researchers then removed the cationic polypeptide fraction from the vaginal fluid and tested the depleted fluid's anti-HIV-1 activity. They found that most of the anti-HIV activity was gone.
Next, the researchers added the cationic polypeptide fraction back into the depleted vaginal fluid, and found that they were able to restore activity against HIV-1.
To further characterize the cationic proteins in vaginal fluid, the researchers used a special two-dimensional gel to separate out proteins in vaginal fluid. The 2D gel was developed especially for separating out cationic proteins. It separates proteins in the first dimension based on cationic charge density, such that smaller, more positively charged peptides migrate faster. In the second dimension, proteins were separated out in tricine-SDS-PAGE a type of electrophoresis gel that allows for the resolution of smaller weight proteins that are below 50 kilodaltons.
Proteins were extracted from protein spots on the gel, digested with trypsin, then analyzed using MALDI TOF/TOF mass spectrometry. In total, 18 cationic polypeptides were identified.
"Some of the proteins were surprising. We hadn't found cystatins or galectin [in vaginal fluid] before," said Cole. "Some proteins, such as lysozyme, defensins, and products from neutrophils, we expected to be there."
Nearly all of the cationic polypeptides identified were either known antimicrobials or had other reported roles in host defense, Cole noted.
Once the cationic polypeptides in vaginal fluid had been identified using a proteomic approach, the researchers decided to test each protein individually to see if it had anti-HIV-1 activity. Interestingly, under physiological concentrations, 13 of the cationic polypeptides showed no anti-HIV-1 activity when tested alone. However, when they were combined, they partially restored the anti-HIV-1 activity in vaginal fluid depleted of cationic polypeptides.
"These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluids," the researchers wrote.
Cole said he would like next to investigate the effects of combining the identified polypeptides with a topical antimicrobial that his lab has been developing for the past four years.
The active ingredient in the topical antimicrobial is a protein called retrocyclin that was found by Cole's research group in 1999 to be active against HIV. The protein was originally isolated from monkeys. It is not made in humans, though humans carry a homologous gene for it.
Currently, the retrocyclin contained in the anti-HIV antimicrobial is artificially synthesized, based upon the human gene sequence for the protein. The synthetic human form of retrocyclin was found to be more effective against HIV than the monkey form, Cole explained.
The retrocyclin-based topical is currently in pre-clinical stages, Cole said. It is the first anti-HIV topical microbicide that is based on natural defense agents, he added.
"Should retrocyclin synergize with proteins in vaginal fluid, that will only help," said Cole. "We are at least expecting that in clinical trials they will work as well as they performed ex vivo in the absence of vaginal fluid."
Cole said he hopes the retrocyclin microbicide will go into pilot clinical studies within the next few years.
Tien-Shun Lee ([email protected])