Reference neutralizing antibody (NA) reagents are needed for laboratories to be able to compare results of neutralization assays that will be used to monitor HIV-1 vaccine recipients. In an effort to establish such reference reagents two asymptomatic, seropositive patients were identified with medium to high amounts of cross-reactive NA activity against a number of HIV-1 strains. Sera obtained from each individual at three or four sequential phlebotomies were pooled, and the two pools were each distributed in > 3000 aliquots into glass ampoules and lyophilized, and the ampoules were flame sealed. An HIV-1 antibody-negative reference serum was prepared in a similar fashion after pooling serum from four individuals. Ampoules were tested for uniformity of fill, sterility, moisture content, residual oxygen, stability, infectivity, and presence of antibody. An international collaborative study was conducted to determine the potency of the samples in six laboratories, each using their own neutralization assays and reagents. The results indicated reasonable consistency between laboratories and that both sera have sufficient titers against a variety of strains for use as reference reagents. These reference sera have been included in the World Health Organization (WHO) AIDS Reagent Project and are available through the three AIDS reagent repositories.

We studied the relationship between early human immunodeficiency virus type 1 (HIV-1) specific immune responses and pathogenesis of infection in participants enrolled in the multicenter AIDS cohort study (MACS). Sera collected at 6-month intervals for 2 years (visit 1-5) from 39 persons who seroconverted by enzyme-linked immunosorbent assay (ELISA) 6 months (visit 2) after enrollment were examined for isotype-specific Western blot reactivity, neutralizing antibodies (NA) against two divergent strains of HIV-1 (HIV-1IIIB and HIV-1RF), and for antibodies capable of participating in antibody-dependent, cell-mediated cytotoxicity (ADCC). These results were compared with changes in CD4+ cell number and episodes of lymphadenopathy. Twenty-five subjects had antibodies of at least one isotype reactive to at least one HIV-1 protein by Western blot at visit 1, before they became ELISA positive. NA reactive with HIV-1IIIB were detected before those reactive with HIV-1RF. NA were first observed in 11 sera at visit 2, in 22 sera at visit 3, and in 3 sera at visit 4; sera from three patients remained nonneutralizing through visit 5. In most cases, NA were detected after a decline in CD4+ cell numbers. The data are consistent with the interpretation that NA develop after about 16 to 18 months of declining CD4+ cell numbers, following which the rate of decline in CD4+ cell numbers slows. In contrast, HIV-1 envelope antigen-specific ADCC responses were first observed in 11 subjects at visit 1 when all 39 were NA and ELISA negative, in 12 subjects at visit 2, in 13 subjects at visit 3, and 1 subject at visit 4. Early ADCC responses were associated with high mean % CD4+ cell numbers and absence of lymphadenopathy throughout the 2-year observation period. Not all subjects who developed ADCC developed NA. In some subjects, ADCC and NA were detectable for the first time at the same visit, for others ADCC was detectable prior to NA, and for a few NA was detectable prior to ADCC. These findings suggest that ADCC and neutralization are mediated by different antibody populations, that they may partially inhibit the progress of HIV-1 infection, and that the late appearance of NA may relate to the failure of immunity to effect recovery from this infection.

The role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and LFA-1 in human immunodeficiency virus type 1 (HIV-1)-induced cell fusion was investigated in subclones of a T-cell leukemic cell line (CEM) with differing abilities to form syncytia. Addition of monoclonal antibodies 84H10 directed against ICAM-1 and MHM23 directed against the common beta subunit of LFA-1 (CD18) resulted in greater than 50% suppression of syncytia formation in cultures of these clones infected with cell-free virus. Two subclones, 2G5-144-84 and 2G5-1, were deficient in their ability to form syncytia and expressed reduced amounts of LFA-1 compared with the parental line. The expression of ICAM-1 but not LFA-1 was upregulated on the clones following treatment with interferon-gamma (IFN gamma); however, this did not overcome the delay in syncytia formation observed in these cells. The syncytia-positive subclones 1B11-39 and 17D-9 expressed high levels of LFA-1. Basal expression of ICAM-1 was upregulated on these cells by treatment with tumor necrosis factor-alpha (TNF alpha), which also accelerated and enhanced syncytia formation. However, anti-ICAM-1 and anti-LFA-1 (CD18) antibodies did not reverse the TNF alpha-induced enhancement of syncytia formation of HIV-1-infected clones 1B11-39 and 17D-9. Under conditions of low viral expression, adhesion molecules may contribute to syncytia formation if adequate levels of both receptor and ligand in the ICAM-1/LFA-1 complex are expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative α-helix region of gp41. A series of chimeras from NS and NRenv genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric envgene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.

The phorbol ester phorbol myristate acetate (PMA) strongly inhibits human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation; it has been suggested that this inhibitory effect is due to the transient downmodulation of the surface-associated CD4 receptors by PMA (I. H. Chowdhury, Y. Koyanagi, S.