The ability of antibodies to neutralize diverse primary isolates of human immunodeficiency virus-type 1 in vitro has been questioned, with implications for the likely efficacy of vaccines. A recombinant human antibody to envelope glycoprotein gp120 was generated and used to show that primary isolates are not refractory to antibody neutralization. The recombinant antibody neutralized more than 75 percent of the primary isolates tested at concentrations that could be achieved by passive immunization, for example, to interrupt maternal-fetal transmission of virus. The broad specificity and efficacy of the antibody implies the conservation of a structural feature on gp120, which could be important in vaccine design.

A panel of anti-gp120 human monoclonal antibodies (HuMAbs), CD4-IgG, and sera from people infected with human immunodeficiency virus type 1 (HIV-1) was tested for neutralization of nine primary HIV-1 isolates, one molecularly cloned primary strain (JR-CSF), and two strains (IIIB and MN) adapted for growth in transformed T-cell lines. All the viruses were grown in mitogen-stimulated peripheral blood mononuclear cells and were tested for their ability to infect these cells in the presence and absence of the reagents mentioned above. In general, the primary isolates were relatively resistant to neutralization by the MAbs tested, compared with the T-cell line-adapted strains. However, one HuMAb, IgG1b12, was able to neutralize most of the primary isolates at concentrations of Յ1 g/ml. Usually, the inability of a HuMAb to neutralize a primary isolate was not due merely to the absence of the antibody epitope from the virus; the majority of the HuMAbs bound with high affinity to monomeric gp120 molecules derived from various strains but neutralized the viruses inefficiently. We infer therefore that the mechanism of resistance of primary isolates to most neutralizing antibodies is complex, and we suggest that it involves an inaccessibility of antibody binding sites in the context of the native glycoprotein complex on the virion. Such a mechanism would parallel that which was previously postulated for soluble CD4 resistance. We conclude that studies of HIV-1 neutralization that rely on strains adapted to growth in transformed T-cell lines yield the misleading impression that HIV-1 is readily neutralized. The more relevant primary HIV-1 isolates are relatively resistant to neutralization, although these isolates can be potently neutralized by a subset of human polyclonal or monoclonal antibodies.

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.

Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin Gl (IgGl) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgGl chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgGl). The 2D10 mouse-human IgGl chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgGl chimeric antibody (chl8B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (ml8B7). chl8B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. chl8B7 and m18B7 enhanced fungistatic or fungicidal activity ofJ774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgGl constant chain can be effective in mediating antifungal activity against C. neoformans. chl8B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis.Immunosuppressed individuals are particularly susceptible to infections with the fungus Cryptococcus neoformans. In recent years the prevalence of cryptococcal infections has increased dramatically because this fungal infection occurs in up to 10% of patients with AIDS (72). In the setting of AIDS C. neoformans infections are particularly difficult to treat because antifungal therapy does not eradicate the infection (67) despite in vitro susceptibility to drugs used in therapy (12).AIDS patients who survive the initial presentation undergo lifelong antifungal suppression therapy to decrease the likelihood of relapse. The difficulty of treating C. neoformans infections in immunocompromised patients has led to interest in the potential of passive antibody therapy as a means to enhance residual cellular immunity (8, 23, 51-53, 55, 63). Limited experience with the use of rabbit polyclonal antibody in humans suggests that the combination of antibody with amphotericin B may improve therapy (29,31,47). Experimental support for a combined approach includes the finding that antibody can potentiate the antifungal effect of amphotericin B in murine models of cryptococcosis (22,30,54).The ability of passive antibody to mediate protection in murine models of C. neoformans infection is dependent on the quantity (23), isotype (53,63), and fine specificity (53) of the antibody reagent and the animal model used (23-25, 35, 53). Antibody to C. neoformans capsular polysaccharide does not inhibit the fungus in the absence of effector cells (24), and antibody-mediated protection is most likely the result of enhanced cellular and nonspecific immune mechanisms (10,19,20,40,42,57,63). Antibodies to the capsular polysacchar...