Linear epitope mapping of humoral responses induced by vaccination with recombinant HIV-1 envelope protein gp160.

To enhance utility of the linear epitope mapping (Pepscan) technique for assay of humoral responses linked to vaccination, two modifications were tested. First, peptides were incubated with serum contained in baths rather than individual wells. Second, a rigorous statistical model was developed to determine which peptide/antibody-binding interactions were significant. The modifications increased the ability to detect signal in these experiments by 15- to 45-fold. These two modifications were applied to linear epitope mapping of HIV seropositive volunteers under treatment with recombinant HIV gp160 and also to rabbits immunized with the same product. Changes in fine specificity of response were observed in animal models and human vaccine recipients over the course of an immunization series with this antigen.

Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.

Mucosal immune responses in four distinct compartments of women infected with human immunodeficiency virus type 1: a comparison by site and correlation with clinical information.

Because mucosal immune responses may be important in protection against human immunodeficiency virus type 1 (HIV-1), HIV-1-specific immune responses at mucosal sites in natural infection were compared. Total antibody concentrations and HIV-1-specific binding antibody responses in four distinct mucosal sites and serum were assessed in 41 HIV-infected and 19 HIV-seronegative women. HIV-1 gp160-specific IgG responses were detected in >99% of mucosal samples in infected subjects, with the highest titers in genital secretions. HIV-1-specific IgA was detected in the majority of endocervical secretions (94%) and nasal washes (95%) but less often in vaginal washes (51%) and parotid saliva (38%). There was no significant correlation between mucosal immune response and most clinical factors. Based on methodologic considerations, frequencies of detection, and HIV-1-specific responses, nasal washes and genital secretions may each provide important measures of HIV-1-specific mucosal immune responses in infected women.

Serum IgA-mediated neutralization of HIV type 1.

The sera of 33 HIV-1-infected individuals, previously shown to neutralize HIV-1MN in vitro, were screened by ELISA for IgA reactivity against rgp120MN and a synthetic V3MN loop peptide. Six were selected for evaluation of the effect of serum IgA from infected individuals on the in vitro infection of susceptible target cells by HIV-1MN. By using protein G immobilized on Sepharose, we depleted the sera of IgG to a level undetectable by nephelometry and viral envelope-specific ELISA. The IgA component of the IgG-depleted serum was affinity purified with immobilized jacalin, a lectin that selectively binds the IgA1 fraction of human Ig. IgG-depleted sera and purified IgA1 serum fractions showing IgA reactivity against rgp120MN and V3MN by ELISA inhibited the in vitro infection of CEM-ss cells by HIV-1MN, but sera depleted of both IgG and IgA1 did not. These data show that, like serum IgG, serum IgA from selected HIV-1-infected individuals is capable of neutralizing HIV-1MN in vitro. The biologic significance of this observation and the identities of serum IgA-recognized HIV-1 neutralization epitopes remain to be determined.

Lytic effects of serum and mononuclear leukocytes on oral epithelial cells in recurrent aphthous stomatitis.

A radioisotope-release assay, utilizing 51Cr-labeled epithelial cells derived from non-keratinizing oral mucosa, was developed to investigate in vitro cytolytic reactions correlating with recurrent aphthous stomatitis (RAS). The cytolytic effects of sera and mononuclear leukocytes from patients in the early stage of ulceration were compared with those from matched RAS-negative control subjects. RAS sera induced significantly more cytolysis than did matched control sera. Heating the RAS or control sera for 30 min at 56 degrees C abrogated their cytotoxic activity. RAS mononuclear leukocytes, like their matched controls, showed no significant direct cytotoxicity. Heat-inactivated RAS or control sera acting in concert with RAS or control mononuclear leukocytes showed no consistent cytolytic effects. However, the heat-inactivated sera of some RAS patients, when combined with autologous mononuclear leukocytes, induced significantly more cytolysis than did either component acting alone. Thus heat-labile humoral factors and, in some cases, mononuclear leukocytes acting in concert with heat-stable serum factors are implicated in RAS-associated in vitro cytolytic reactions. These findings suggest that the effector mechanisms of such reactions include both complement-mediated and antibody-dependent cell-mediated cytotoxicity.