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The major route of transmission of the human immunodeficiency virus type 1 (HIV-1) worldwide, like other agents of sexually transmitted disease, is via mucosal surfaces of the genital tract through sexual exposures (1-4). It has been hypothesized that immune responses at these sites may be important determinants of protection against HIV-1 (1,2,5). Development of HIV-1 vaccines which elicit local mucosal immune responses may be critical for an effective preventive vaccine. Specific mucosal immune responses elicited after different routes of mucosal immunization in rodents have been examined previously (6-16). Nasal and oral immunization routes have elicited strong mucosal responses to HIV-1 peptides and whole proteins in mice (7,11,17,18,24). Induction of mucosal and systemic immunity to SIV and HIV-1 antigens in primates has also been obtained using a variety of mucosal routes of vaccine administration (19-22). [ABSTRACT FROM AUTHOR]

The major route of transmission of the human immunodeficiency virus type 1 (HIV-1) worldwide, like other agents of sexually transmitted disease, is via mucosal surfaces of the genital tract through sexual exposures (1-4). It has been hypothesized that immune responses at these sites may be important determinants of protection against HIV-1 (1,2,5). Because of the potential relationship of mucosal immune responses to protection against HIV-1 infection, an assessment of immune responses at these sites in vaccine trial participants will be critical. Proper collection and processing of specimens from mucosal sites is of vital importance to the measurement of mucosal immune responses. This chapter details the collection and processing procedures of specimens from three distinct mucosal compartments in female subjects. Collection techniques for additional mucosal compartments (e.g. semen, rectal, etc.) can be found in ref. 6 (see Note 1). [ABSTRACT FROM AUTHOR]

B-cells play an important role in protection against pathogens, and they secrete specific antibodies in serum and mucosal secretions upon antigenic stimulation contributing to immune exclusion and clearance of pathogens. The frequency of antibody forming cells (AFC) in specific organs is often a reflection of the route of antigen exposure, i.e., systemic, oral, or intranasal, as well as of antigenic load. Enumeration of AFC was originally performed by plaque-forming cell assay measuring lysis of sheep red blood cells. The nature of this assay, that requires coupling of the antigen to sheep red blood cells (SRBC), made detection of various antigen-specific AFC rather cumbersome. However, the development of the enzyme linked immunodetection of AFCs (ELISPOT) combined with the development of standardized lymphocyte isolation techniques enables detection of AFC secreting antibodies specific for many different antigens and derived from various immunologic effector sites (1-6). [ABSTRACT FROM AUTHOR]

Accurate analyses of antibody specificities and affinities are critical to understanding their role in antibody-mediated neutralization of HIV-1 in vitro and their potential activity against HIV-1 in vivo. Multiple immunologic tools currently exist for measuring antibody-antigen interactions to include enzyme immunoassays (EIA), Western blotting, radioimmunoassays, and others. However, these techniques all require some form of protein labeling or secondary antibodies for detection of specific binding interactions. Disadvantages of protein labeling involve potential alterations in protein tertiary structure, while the use of secondary detection antibodies prohibits the measurement of binding interactions in real-time. Recently, instruments utilizing the physical principal of surface plasmon resonance (SPR) (BIAcore™, Pharmacia Biosensor, Piscataway, NJ) (1-4) have been developed which allow real-time measurements of protein binding interactions without the use of internal labels or secondary antibodies. Proteins of interest are covalently coupled to a flexible biosensor matrix, and kinetic rate constants can be directly extracted from analyses of association and dissociation binding curves. The relative concentration of immobilized protein can be determined allowing, for example, the comparison of binding efficiency of one antibody against a number of different proteins. [ABSTRACT FROM AUTHOR]

Protective antibody responses against HIV-1 have yet to be identified or determined. HIV-1 envelope gpl20/gp41 is known to exist as a multimer (tetramers or trimers) on the surface of the virion (1-4). A number of immunoassays have been developed to evaluate HIV-1-specific binding antibody responses using peptides, fusion proteins, and recombinant proteins. Attempts to correlate antibody binding parameters with in vitro HIV-1 neutralization capacity have identified correlations between the presence of V3 antibodies and the capacity to neutralize T-cell line adapted strains of HIV-1. However, no correlation with neutralization of primary HIV-1 isolates and v3 antibodies have been identified (5). Furthermore, binding to monomeric forms of gpl20 has shown little correlation with neutralization of the homologous primary HIV-1 indicating that epitope accessibility and tertiary structure of monomeric forms of gpl20 differs from quaternary structure of membrane expressed oligomeric gpl20/gp41 (6-8). [ABSTRACT FROM AUTHOR]