SUMMARYMurine MoAbs to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) polysaccharide are protective in mice in vivo and in vitro. The prevalence of protective anti-GXM antibodies in human serum is unknown. To provide further insight into the human antibody response to C. neoformans we determined the prevalence, isotype, and IgG subclass utilization of human anti-GXM antibodies in HIV+ and HIV-sera by a sensitive antigen capture ELISA assay. One hundred and twenty-three sera from the Bronx Municipal Hospital Centre serum bank were studied retrospectively. Seventy were from HIV+ individuals, 10 with a history of cryptococcal meningitis (CM), and 53 were from HIV-individuals. Serum GXM determinations were also performed on 61 HIV+ sera. Our results demonstrated that anti-GXM IgG, IgA, and IgM are ubiquitous in both HIV+ (including those with CM), and HIV-sera. Anti-GXM IgA titres and total serum IgA concentration were elevated in HIV+ sera. Anti-GXM IgG antibodies were almost exclusively isotype-restricted to the IgG2 subclass. Our data also demonstrated elevations of antibovine serum albumin (BSA) titres in HIV+ sera. Taken together, our findings confirm hypergammaglobulinaemia and expansion of anti-protein (BSA) antibodies in HIV+ individuals and isotype restriction of human anti-carbohydrate (GXM) antibodies to the IgG2 subclass. Our report of ubiquitous anti-GXM antibodies of the IgG and IgA isotypes suggests that anti-GXM antibodies exist before HIV infection.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy-and light-chain immunoglobulin variable region (V. and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb El originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the El, VH uses V,1 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a V,Ser-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses V,5.1 and J,1. In MAbs 1255 and 339, the V. uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a V.21-like gene element and J.5. In MAbs 302 and 386, respectively, the VH uses V,GAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses V,4/5-like gene elements and J,1. VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confinn that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding. Cryptococcus neofornans is an encapsulated fungus that causes life-threatening meningoencephalitis in up to 9% of patients with AIDS (53). Treatment of cryptococcal meningitis in patients with AIDS is difficult because antifungal drugs often

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The Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) has been conjugated to tetanus toxoid (GXM-TT) as an investigational vaccine. GXM-TT elicits antibodies that are protective in C. neoformans-infected mice. In an effort to characterize the fine specificity and molecular structure of human GXM-TT-elicited antibodies, we generated two GXM monoclonal antibodies (MAbs) from peripheral blood lymphocytes of a volunteer GXM-TT recipient and studied serum GXM antibody idiotype expression in 10 additional vaccinees. The MAbs, 2E9 and 3B6, are the immunoglobulin M(lambda) isotype and bind capsular polysaccharides of C. neoformans serotypes other than the serotype A that was used for immunization. Neither antibody competes with murine GXM MAbs for antigen binding, suggesting that the human MAbs recognize a different epitope. The B-cell superantigen staphylococcal protein A binds both MAbs, and human immunodeficiency virus gp120 binds 2E9. MAb nucleic acid sequence analysis revealed that both antibodies use an identical V lambda 1a-J lambda genetic element with different, somatically mutated, members of the VH3 gene family and different DH and JH gene elements. The gene elements used by both MAbs occur in fetal B-lymphocyte repertoires, autoantibodies, and other polysaccharide antibodies. Post-GXM-TT vaccination GXM antibodies from 10 additional vaccinees expressed a shared idiotype defined by rabbit antiserum raised against MAb 2E9. Our data suggest that the human GXM antibody response is restricted and raise questions regarding the importance of specific variable-region elements and superantigens in the generation of human antibody responses to encapsulated pathogens.

We studied the rates of and factors associated with self-reported prior human immunodeficiency virus (HIV) testing in adult patients visiting an emergency department (ED) in the Bronx, New York City. A total of 1,744 consecutive noncritical adult medical emergency patients responded to a standardized interview administered by ED physicians. The interview included questions pertaining to demographic characteristics, prior HIV testing, and HIV risk behaviors. On multivariate analysis, female gender, younger age, history of weight loss, injecting drug use (IDU), syphilis, and genital herpes were all associated with increased reported prior testing rates. Race (i.e., black race) was an independent predictor of increased rates among male subjects; comparatively low rates were reported by patients with a first language other than English, patients lacking medical insurance, and highly sexually active, nonblack men. Increased HIV testing rates were reported by subjects with recognized HIV risk behaviors in a New York City ED population; however, substantial proportions of subjects at risk had not been tested. Programs of HIV testing and counseling need to include older, uninsured, and non-English-speaking segments of the population who engage in high-risk behaviors.