Human cervicovaginal mucus contains an activity that hinders HIV-1 movement.

Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.

Transferrin conjugation confers mucosal molecular targeting to a model HIV-1 trimeric gp140 vaccine antigen.

The generation of effective immune responses by mucosal vaccination without the use of inflammatory adjuvants, that compromise the epithelial barrier and recruit new cellular targets, is a key goal of vaccines designed to protect against sexually acquired pathogens. In the present study we use a model HIV antigen (CN54gp140) conjugated to transferrin (Tf) and evaluate the ability of the natural transferrin receptor CD71 to modulate immunity. We show that the conjugated transferrin retained high affinity for its receptor and that the conjugate was specifically transported across an epithelial barrier, co-localizing with MHC Class II(+) cells in the sub-mucosal stroma. Vaccination studies in mice revealed that the Tf-gp140 conjugate elicited high titres of CN54gp140-specific serum antibodies, equivalent to a systemic vaccination, when conjugate was applied topically to the nasal mucosae whereas gp140 alone was poorly immunogenic. Moreover, the Tf-gp140 conjugate elicited both IgG and IgA responses and significantly higher gp140-specific IgA titre in the female genital tract than unconjugated antigen. These responses were achieved after mucosal application of the conjugated protein alone, in the absence of any pro-inflammatory adjuvant and suggest a potentially useful and novel molecular targeting approach, delivering a vaccine cargo to directly elicit or enhance pathogen-specific mucosal immunity.

Microbicides for HIV/AIDS. 3. Observation of apparent dynamic protonation and deprotonization in CD4+ T-cell model systems.

New measurements of the electrophoretic mobility of T-cell model systems have been carried out and analyzed to obtain the dynamic variation in mobility in small titration increments during separate upscale and downscale sweeps in pH. We demonstrate that a plot of plambda vs p[NaCl] has been found essential in evaluating the consistency of electrophoretic mobility measurements at different (1:1) electrolyte concentrations and show, for the first time, that electrophoretic mobility measurements as a function of pH can reflect different rates of the respective ionization and association that occur in the surface functional groups as a consequence of the different changes in the hydration-dehydration reactions involved. Differences found between the upscale and downscale sweeps suggest that it is easier to protonate a protein cell surface than to deprotonate it. The effect is most pronounced at the highest salt concentration (similar to that which exists for the cells in their native state) and becomes less pronounced as the salt concentration is lowered. The effect is interpreted as a result of the different changes in the state of hydration as a proton moves from the bulk through the double layer to a surface group and the reverse. The effect occurs with both replicating and activated T-cells. This latter result may be of biological significance and particularly relevant to HIV-1 infection, since during male-to-female transmission, the environment where most infections occur supports this protonation effect.

Microbicides for HIV/AIDS. 2. Electrophoretic fingerprinting of CD4+ T-cell model systems.

New measurements of the dependence of the surface charge on the pH and electrolyte concentration for three living human white blood cell lines that are the principal targets of the HIV-1 virus are reported. Comparison of the electrophoretic fingerprint (EF) pattern, especially the line of zero mobility, with that of reference colloids establishes the separate individual identities and shows that all three exhibit a zwitterionic surface. With the EF results as a guide, preliminary biological infectivity measurements showed that small polyvalent cations modulate the negative charge on the T-cell surface in a way that strongly affects the infection kinetics. H9 cells were exposed to an infectious virus (X4), and the data showed that HIV interaction with target cells is enhanced by physiological fluids. The nondestructive methodology described is generally applicable to characterization of the surface charge and determination of the colloidal stability of any aqueous charged colloidal system without reference to any model of the double layer.