Antibodies of symptomatic human immunodeficiency virus type 1-infected individuals are directed to the V3 domain of noninfectious and not of infectious virions present in autologous serum

Abstract

The present study was designed to determine the antibody specificity for the human immunodeficiency virus type 1 (HIV-1) V3 domains of infectious and noninfectious virions present in the serum of AIDS patients. To accomplish this, HIV-1 was isolated in the presence of autologous antibodies from the serum samples of six AIDS patients in HIV-1-negative donor peripheral blood mononuclear cells by short-term cultivation. The isolated virus, defined as the infectious cell-free virus (iCFV), was characterized by sequence analysis of the proviral DNA coding for the third hypervariable (V3) region of the external glycoprotein gpl20. This was carried out by amplifying and cloning the V3 region. In all six cases studied, 20 randomly selected V3 clones derived from the proviral DNA of the iCFV, 20 clones from patient cell-free virus, and 20 clones from cell-integrated virus were sequenced to study the distribution and frequency of the intrapatient virus population. The number of major virus variants in the six patients ranged from three to nine. The various V3 sequences found in the AIDS patients showed the typical amino acid pattern of the syncytium-inducing and non-syncytium-inducing viral phenotypes characteristic for the late stage of infection. However, only one patient-specific iCFV variant was detected within the 20 V3 clones analyzed per virus isolation. For the six patients a total of 34 V3-loop variants, either iCFV or non-iCFV, was observed. All 34 V3-loop sequences were expressed as glutathione-S-transferase fusion proteins (V3-GST). The autologous antibody response to the V3-GST fusion proteins was studied by Western immunoblot analysis. A strong antibody response to almost all non-iCFV V3-GST proteins was found in the sera of the six patients. In contrast, the autologous antibody response to the six iCFV V3 loops was undetectable (in four patients) or very faint (in two patients) compared with that to the non-iCFV V3 loops. Five of the six iCFV loops showed positively charged amino acids at positions strongly associated with the syncytium-inducing phenotype. These findings suggest that our in vitro isolation system selects for virions which are not recognized by V3-specific antibodies and are infectious both in vitro and in vivo.

Escape of HIV-1 is associated with lack of V3 domain-specific antibodies in vivo

“…This high specificity was not observed with short peptides spanning the identical V3 sequences (pep-scan analysis, data not shown). Thus the synthesis of the full-length V3 loop is needed to detect differences in the V3 antibody response [21,22]. In humans, progression of the disease was shown to correlate with env-directed or V3-directed homologous antibodies [29] or with loss of neutralizing antibody titres [30][31][32].…”

“…For polymerase chain reaction (PCR) analysis patients' PBMC were isolated by Ficoll-Paque gradient centrifugation. The iCFV was isolated by culturing 1 × 10 6 stimulated donor PBMC (same group of donors) in 20% serum freshly collected from patients F and G. Nested PCR of cultured or patient-derived PBMC, reverse transcriptase (RT)-PCR of cell-free viral RNA, the cloning and sequencing, and the GST-V3 ELISA were carried out as described previously [21]. In brief, donor PBMC were infected in vitro with the patient's serum.…”

“…In a previous study [21] we reported that in AIDS patients the infectivity of CFV was closely related to the lack of type-specific antibodies directed against the V3 domain of the so-called iCFV. All other V3 variants simultaneously identified in the patients were unable to infect donor peripheral blood mononuclear cells (PBMC), and were therefore designated as non-iCFV.…”

Effects of mutations on HIV‐1 infectivity and neutralization involving the conserved NNNT amino acid sequence in the gp120 V3 loop

“…These primary viruses showed an atypically mutated g15 region where 3-4 of 4 amino acids were replaced (NNNT amino acids, 300-303, Fig 1A). However, such atypical mutations have also been identified in other primary isolates [22] or are present in V3 sequences published by the HIV sequence database [11]. As mentioned before, mutations within the V3 loop affect the usage of coreceptors, antibody binding and N-glycosylation.…”

Enhancing of anti-viral activity against HIV-1 by stimulation of CD8+ T cells with thymic peptides

“…The isolation of cell-free neutralization-resistant patient isolates from the serum of AIDS patients has been described before [24]. Virus stocks were grown in peripheral blood mononuclear cells (PBMC) and used for NSI and SI testing and neutralization assays in PBMC.…”