Maternal monoclonal antibody to the V3 loop alters specificity of the response to a human immunodeficiency virus vaccine.

The effect of maternally transferred monoclonal antibody (MAb) on the offspring antibody response to rgp120SF2 was examined in a murine model. Two MAbs were studied: MAb 83.1, which recognizes a determinant in the V3 loop of gp120 from human immunodeficiency virus-1 (HIV-1) SF2, and MAb 26.2D3, which recognizes a conserved N-terminal region of gp120 from HIV-1SF2. Offspring were immunized at 18-21 days of age with 100 micrograms of rgp120SF2 in complete Freund's adjuvant. Offspring immunized in the presence of preexisting MAb 83.1 but not MAb 26.2D3 demonstrated inhibition of the IgG anti-V3 response. The total IgG anti-rgp120SF2 response was not affected by preexisting MAb. Since newborns at risk for HIV may be immunized in the presence of maternal or administered anti-HIV antibody, alternative strategies may be required to circumvent inhibition of the infant's epitope-specific response to HIV immunization by preexisting antibody.

Inhibition of the offspring anti-recombinant gp120 antibody response to a human immunodeficiency virus vaccine by maternal immunization in a murine model.

A murine model was developed for assessing the effects of passively transferred polyclonal maternal anti-gp120 antibodies on the subsequent immunization of the offspring with recombinant gp120SF2 in complete Freund's adjuvant (rgp120SF2-CFA). Adult female BALB/c mice were immunized with rgp120SF2-CFA 6 weeks before mating. The 3-week-old offspring were subsequently immunized with the same vaccine and followed for 9 weeks. Both the total IgG anti-rgp120SF2 and the anti-V3 IgG antibody response to vaccine were inhibited in the experimental animals. The total IgG anti-rgp120SF2 response was < 20% of the control response (P < .001) 9 weeks after immunization. Anti-V3 antibody was also decreased. As vaccine studies begin in infants, the effects of preexisting antibody on the infant response to human immunodeficiency virus vaccines must be considered.

Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons.

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.

Sequential immunizations with rgp120s from independent isolates of human immunodeficiency virus type 1 induce the preferential expansion of broadly crossreactive B cells.

The gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is a dominant target against which the host's humoral immune response is directed. Unfortunately, gp120 proteins from different isolates of HIV are antigenically distinct, complicating the use of the envelope glycoprotein in vaccines designed to prevent acquired immunodeficiency syndrome. Using an enzyme-linked immunosorbent spot assay (ELISA), BALB/c mice immunized and boosted with recombinant purified gp120 were studied at the single cell level for their humoral immune response to HIV-1 envelope proteins. Approximately 90% of responding B cells produced antibodies reactive with the immunizing form of gp120 but not with gp120s from other strains of HIV. A novel sandwich ELISA was then used to analyze the frequency with which individual in vivo activated B cells produced antibodies that crossreacted with heterologous gp120s. Repeated immunizations with a single gp120 or with a mixture of different gp120s resulted in the activation of primarily mono-specific (noncrossreactive) B cells. In contrast, the sequential immunization of mice with recombinant purified envelope proteins from different strains of HIV (IIIB, SF2, and Zr6) induced the selective expansion of B cells producing highly crossreactive antibodies.

Importance of hypervariable regions of HIV-1 gp120 in the generation of virus neutralizing antibodies.

Variants of the envelope gene of the HIV-SF2 isolate of HIV-1 with deletions of one or more of the hypervariable domains of gp120 were produced in genetically engineered yeast as nonglycosylated denatured polypeptide analogs of gp120. Purified antigens were used to immunize experimental animals to determine whether the removal of hypervariable regions from this type of gp120 immunogen had any effect on (1) the ability of the antigen to elicit virus neutralizing antibodies; and (2) the isolate specificity of the neutralizing antibodies that were elicited. The results of these studies demonstrate that, in addition to the previously identified V3 domain, at least two other hypervariable regions in gp120 are capable of eliciting neutralizing antibodies in experimental animals. However, when all five of the hypervariable regions were deleted, the resulting antigen was no longer capable of eliciting neutralizing antibodies. Finally, the neutralizing antibodies elicited by all of these nonglycosylated antigens were effective against HIV-SF2, the isolate from which the antigens were derived, but were not able to neutralize two divergent isolates, HIV-BRU or HIV-Zr6.

Recombinant polypeptide from the endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals.

Sera from the majority of individuals that were positive in an enzyme-linked immunosorbent assay (ELISA) retrovirus (ARV), an isolate of the for antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV), an isolate of the retrovirus identified as the etiologic agent of AIDS, were found to react with a 31,000-dalton protein (p31) in virus Western blot assays. To determine if this 31,000-dalton immunoreactive species originated from the putative endonuclease region of the polymerase (pol) gene of ARV, we cloned this portion of pol into bacterial expression vectors for direct expression and for expression as a fusion protein with human superoxide dismutase. Transformants from both constructions expressed immunoreactive protein detected in immunoblots with an AIDS patient's serum. Extracts from transformants expressing these sequences competed with the binding of antibodies from AIDS patients' sera to the 31,000-dalton protein in virus immunoblots, confirming that viral p31 originated from the endonuclease domain of the ARV polymerase gene. The superoxide dismutase-p31 fusion protein was purified, and an ELISA for detecting antibodies to p31 was developed. The majority (95%) of serum samples obtained from individuals seropositive in the virus ELISA were also positive in the p31 antibody ELISA.

Simultaneous determination of total IgE and allergen-specific IgE in serum by the MAST chemiluminescent assay system.

We have developed a chemiluminescent immunoenzymometric system. The first commercial application of this chemiluminescent assay (CLA) is the measurement of total IgE and allergen-specific IgE in human serum. The CLA system is a second-generation adaptation of the MAST RIA allergy profiling system. The MAST CLA system assay protocol consists of three steps: overnight incubation of serum, a 4-h incubation with enzyme-labeled antibody, and a 30-min chemiluminescent reaction, which produces a visible image (immunograph) on high-speed Polaroid instant film. The densities of the bands produced on the film are quantified with an inexpensive microprocessor-controlled infrared transmittance densitometer. The novel luminogenic substrates used yield a constant light output for over 2 h with an intensity at least 10-fold greater than that of commercial chemiluminescent reagents. The MAST CLA system exhibits sensitivity, specificity, and precision equal to that of the MAST RIA system (r = 0.96 for 40 serum samples analyzed with 25 allergens). As many as 35 different allergens per sample can be quantified in a single assay. The MAST CLA system requires no standard curve or volume-dependent pipetting steps, incorporates both positive and negative controls for each sample, and quantifies allergen-specific IgE at picomolar concentrations.