Gp41 Immunodominant Region Research Articles

Conformational plasticity of the HIV-1 gp41 immunodominant region is recognized by multiple non-neutralizing antibodies

The early humoral immune response to acute HIV-1 infection is largely non-neutralizing. The principal target of these antibodies is the primary immunodominant region (PID) on the gp41 fusion protein. The PID is a highly conserved 15-residue region displayed on the surface of HIV-1 virions. In this study, we analyzed the humoral determinants of HIV-1 gp41 PID binding using biophysical, structural, and computational methods. In complex with a patient-derived near-germline antibody fragment, the PID motif adopts an elongated random coil, whereas the PID bound to affinity-matured Fab adopts a strand-turn-helix conformation. Molecular dynamics simulations showed that the PID is structurally plastic suggesting that the PID can form an ensemble of structural states recognized by various non-neutralizing antibodies, facilitating HIV-1 immunodominance observed in acute and chronic HIV-1 infections. An improved understanding of how the HIV-1 gp41 PID misdirects the early humoral response should guide the development of an effective HIV-1 vaccine.

Assessment of Antibody Interference of Enfuvirtide (T20) Function Shows Assay Dependent Variability.

Background:During HIV infection, fusion of the viral and cellular membranes is dependent on folding of the gp41 trimer into a six-helix bundle. Fusion inhibitors, such as the antiretroviral Enfuvirtide (T20), interfere with the formation of the gp41 six-helix bundle. Recent in vitro studies reveal that the gp41 immunodominant region one targeting antibody 3D6 can block T20 interference, but the clinical and pathophysiologic significance of this finding is unclear.Objective/Method:We have previously characterized a number of antibodies that target conformational epitopes on gp41and herein characterized their ability to interfere with T20 in multiple assays and assess their prevalence in HIV infected subjects.Results:The T20 interference by antibody 3D6 was confirmed in a CHO-HXB2 envelope/ HeLaT4+ cell culture assay. Antibodies that target an immunodominant region one epitope, as well as a gp41 discontinuous epitope, also interfered in this assay, however, not all antibodies that targeted these epitopes showed T20 interference. This response was not due to the direct binding of T20 by the antibodies and could not be replicated utilizing TZM-bl and HL2/3 cells. Notably, serum competition studies on a panel of HIV subjects demonstrate that these conformational targeting antibodies are common in the HIV population.Conclusion:The relatively common nature of antibodies targeting these epitopes, the disparate in vitro results, and lack of reported clinical failures ascribed to such antibodies leads us to conclude that antibody interference of T20 is likely not clinically relevant. However, this warrants continued consideration with the advancement of other fusion inhibitors.

Computational analysis of antibody dynamics identifies recent HIV-1 infection.

Accurate HIV-1 incidence estimation is critical to the success of HIV-1 prevention strategies. Current assays are limited by high false recent rates (FRRs) in certain populations and a short mean duration of recent infection (MDRI). Dynamic early HIV-1 antibody response kinetics were harnessed to identify biomarkers for improved incidence assays. We conducted retrospective analyses on circulating antibodies from known recent and longstanding infections and evaluated binding and avidity measurements of Env and non-Env antigens and multiple antibody forms (i.e., IgG, IgA, IgG3, IgG4, dIgA, and IgM) in a diverse panel of 164 HIV-1-infected participants (clades A, B, C). Discriminant function analysis identified an optimal set of measurements that were subsequently evaluated in a 324-specimen blinded biomarker validation panel. These biomarkers included clade C gp140 IgG3, transmitted/founder clade C gp140 IgG4 avidity, clade B gp140 IgG4 avidity, and gp41 immunodominant region IgG avidity. MDRI was estimated at 215 day or alternatively, 267 days. FRRs in untreated and treated subjects were 5.0% and 3.6%, respectively. Thus, computational analysis of dynamic HIV-1 antibody isotype and antigen interactions during infection enabled design of a promising HIV-1 recency assay for improved cross-sectional incidence estimation.

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus (SIV) vaccine enhances protection against neutralization-resistant mucosal SIV infection.

Here, we show that a CD40L-adjuvanted DNA/modified vaccinia virus Ankara (MVA) simian immunodeficiency virus (SIV) vaccine enhances protection against a pathogenic neutralization-resistant mucosal SIV infection, improves long-term viral control, and prevents AIDS. Analyses of serum IgG antibodies to linear peptides of SIV Env revealed a strong response to V2, with targeting of fewer epitopes in the immunodominant region of gp41 (gp41-ID) and the V1 region as a correlate for enhanced protection. Greater expansion of antiviral CD8 T cells in the gut correlated with long-term viral control.

Diversity within the Immunodominant Epitopes of Envelope gp41 HIV-1 in Kenya and Its Effects on Performance of the HIV-1 Antibody-Based Detection Kits

Background: Human Immunodeficiency Virus (HIV) is characterized by high rates of genetic variability in vivo that could affect the performance of the HIV-1 Antibody-Based Detection Kits in use in Kenya. Objective: This study aimed at establishing the diversity of Envelope gp41 Epitopes and its effects on performance of the currently used HIV diagnostic kits in Kenya. Methods: Two hundred (200) HIV positives and 200 HIV negatives samples were collected from the Regional Blood Transfusion Centers (RBTCs) in Kenya. Viral RNA was extracted from 96 HIV Positive samples and sequenced on gp41-immunodominant region (IDR).The sequences obtained were analyzed using various applications in the Los Alamos HIV Database. The HIV gp41-IDR consensus sequence generated was used to synthesize gp41 IDR peptide. The Global HIV gp41-IDR Consensus nucleotide sequence was obtained from literature and used also to synthesis corresponding gp41 IDR peptide. The two peptides were used to prepare HIV Testing ELISA kits. The400 plasma samples that had been collected from this study from RBTCs were tested using five HIV testing kits approved for use in Kenya. The same samples were tested using the two ELISA system developed. Results: The HIV Consensus gp41-IDR peptide from Kenya displays a similarity of 93.0% against HXB2 sequence and 95.3% against Global HIV Consensus gp41-IDR amino acid sequence. There were 331 (7.8%) substitutions in the Consensus gp41-IDR peptides (Kenya) out of which 151substitutions were due to the substitution in positions A96→N (n = 79) and A101→S (n = 75). Both Consensus gp41-IDR peptides (Kenya) and Consensus gp41-IDR peptides (Global) showed common substitutions rate of 4.2% (n = 151). All the kits that were used showed 100% agreement in results. Conclusion: Although the HIV Consensus gp41-IDR peptides (Kenya) showed marked substitutions in respect to Consensus gp41-IDR peptide (Global) there was no difference in effects on the performance of the HIV-1 Antibody-Based Detection Kits in use in Kenya.

P-D3 Structural definition of ADCC epitopes within the gp41 immunodominant region

P-D3 Structural definition of ADCC epitopes within the gp41 immunodominant region

C1 Evaluation of Novel HIV Envelopes in Rabbits Immunized With a DNA Prime/Protein Boost Vaccine Regimen

Background We formerly constructed several DNA plasmids encoding HIV-1Ba-L gp160 containing point mutations and truncations in the cytoplasmic tail of gp41. In addition, we generated plasmid DNA encoding a V2/V3 mutant gp145 and the homologous, purified protein. We hypothesized that immunization of rabbits with these novel antigens, delivered in a DNA prime/protein boost regimen, would result in antibody responses of greater magnitude and breadth. We further hypothesized that immunization of rabbits, with a noted defect in B cell development and antibody selection, may give rise to neutralizing antibodies that would otherwise be removed due to their recognition of self proteins, lipids or long CDR3s. Methods A rabbit immunogenicity study was performed to assess antibody responses elicited in New Zealand White (NZW) rabbits. For this, plasmid DNA encoding either gp160-Q708 (Q708) or gp145-V2/V3mut (V2/V3mut) was administered, by electroporation (Innovio), at weeks 0, 4 and 8. DNA–primed rabbits were then boosted, by intramuscular route, with ribi- (MPL, TDM, CWS)-adjuvanted recombinant protein (gp145-V2/V3mut) at weeks 22, 28 and 38. A similar schedule of DNA and protein immunizations was performed in an SLE (lupus) rabbit model system (a generous gift from Dr. Rose Mage, NIH). Binding antibody titers were assessed by ELISA and neutralizing antibody screens were conducted using a Tzm-b1 pseudo-virus assay. B-cell epitope mapping was performed, by pepscan ELISA, using 20mer peptides, overlapping by 14 amino acids. Results NZW rabbits, immunized with plasmid DNA expressing Q708 or V2/V3mut, generated robust anti-gp145 titers following 3 administrations. Priming with Q708 yielded greater antibody titers (approx. 1 log) compared to V2/V3mut. Antibody titers in DNA-primed rabbits were further augmented following protein boost immunizations with adjuvanted-V2/V3mut protein. Following boost immunization, antibody titers were found to be comparable in both DNA-primed groups. By contrast, anti-gp145 antibody titers were found to be generally less in lupus rabbits following both DNA and protein immunizations. Antibody responses to 2F5/4E10 epitopes were detectable following DNA immunization of NZW rabbits in both (Q708, V2/V3mut) DNA-primed groups. Overall, antibody responses to 2F5/4E10 were comparable in lupus rabbits relative to that noted in NZW rabbits. Following protein boost immunization, 2F5/4E10 antibody responses were only found to be augmented in select rabbits (one from NZW group, one from lupus) that received the V2/V3mut homologous prime/boost regimen. Epitope mapping revealed that protein-boosted rabbits primed with Q708 DNA generated antibody responses to the C1, V2, V3, V5, C5 and immuno-dominant region of gp41. In contrast, protein-boosted rabbits primed with V2/V3mut DNA elicited antibody responses predominantly to the C1, V3, V5, C5 and HR2/MPER regions of gp41. Epitope responses were generally weaker in the V2/V3mut-primed group compared to the Q708-primed group. More robust antibody responses were found when immunizations were performed in lupus rabbits compared to NZW rabbits. In addition to epitope responses found in NZW rabbits, lupus rabbits generated additional antibody responses to the V1 and C4 regions (Q708-primed group), and against the V1, V2, C2/V3, C3, C4 and HR1 regions (V2/V3mut primed group). A neutralization screen found that the sera of immunized rabbits could neutralize a tier 1 (BaL. 1a) but not tier 2 (US1) viral isolate. Enhanced neutralization was not observed in lupus rabbits as compared to NZW rabbits. The greatest inhibition was noted in NZW rabbits primed with Q708 DNA and boosted with V2/V3mut protein. Conclusions These findings suggest that a DNA prime/protein boost immunization strategy incorporating novel Env immunogens with c-tail truncations and V2/V3 loop mutations can elicit robust antibody responses that can neutralize a type-specific viral isolate. While increased breadth of epitope responses was acheived by immunizing lupus rabbits, increased neutralization was not observed against the Bal.1a or US1 isolate tested. Further studies are warranted to investigate if such changes incorporated into other clade B and clade C envelopes will increase binding and neutralizing antibody responses.

Studies on the mechanism of complement-mediated inhibition of antibody binding to HIV gp41.

We have previously demonstrated that HIV envelope gp41 binding to specific antibodies decreases after preincubation of fluid-phase gp41 in normal human serum. This inhibition is proven to be mediated by the classical complement pathway. In this study recombinant gp41 (rgp41) and/or synthetic peptides were preadsorbed to solid phase, and then complement (normal human serum/heated human serum/purified Clq/heated Clq) and anti-gp41 antibodies were added either after each other or simultaneously, and the amounts of bound antibody, and deposited C3b, C4b and Clq were measured. Complement-dependent inhibition of antibody binding to solid-phase rgp41 was found, and Clq seems to be at least partially responsible for this phenomenon. Heating of Clq did not affect this process. Higher amounts of anti-gp41 antibodies significantly and dose-dependently enhanced C4b and C3b fixation to solid-phase rgp41. In the case of synthetic peptides corresponding to the immunodominant region of gp41, significant antibody binding to the solid-phase peptides was also detected, and pretreatment of peptides preadsorbed to solid phase with normal human serum almost totally abolished the antibody binding.

A Bispecific Antibody Composed of a Nonneutralizing Antibody to the gp41 Immunodominant Region and an Anti-CD89 Antibody Directs Broad Human Immunodeficiency Virus Destruction by Neutrophils

In addition to the direct neutralization of virus, there is a broader potential for antibody-mediated inhibition of human immunodeficiency virus (HIV) by targeting HIV to effector cells. We demonstrate here that a bispecific antibody incorporating a broadly reactive anti-gp41 antibody, F240, and an anti-IgA receptor (CD89) antibody is effective at directing neutrophils to destroy HIV. Not only are neutrophils the predominant type of white blood cells and very efficient at mediating cell cytotoxicity, they are relatively resistant to infection with HIV. Therefore, they represent a significant weapon against infection if they can be directed and armed to destroy HIV and infected cells.

High-throughput, functional screening of the anti-HIV-1 humoral response by an enzymatic nanosensor

The impact of antibodies on the target's epitope conformation is a major determinant of HIV-1 neutralization and a potential contributor to disease progression. We explore here a conformation-sensitive enzymatic nanosensor for the high-throughput functional screening of human anti-HIV-1 antibodies in sera. When displaying a model epitope from a gp41 immunodominant region (Env residues from 579 to 613), the sensing signal quantitatively distinguishes between adaptive and non-adaptive antibody binding. By using this tool, we have identified IgG4 as the immunoglobulin subpopulation most efficient in the structural modification of the target epitope.

The first B/G intersubtype recombinant form of human immunodeficiency virus type 1 (HIV-1) identified in Germany was undetected or underquantitated by some commercial viral load assays

The high level of genetic diversity of human immunodeficiency virus type 1 (HIV-1) and the continual emergence of recombinant forms have important implications not only for the global evolution of HIV but also for diagnosis, monitoring, and treatment strategies. The present study reports the first intersubtype B/G recombinant strain of HIV-1 in Germany. This strain is notable from a clinical perspective, since it was undetectable in the NucliSens HIV-1 QT assay (Organon Tecknika/bioMérieux) and was significantly underquantitated in the Monitor v1.5 test (Roche Molecular Systems) relative to the LCx HIV RNA Quantitative assay (Abbott Laboratories). Gag-encoded p24 (gag p24), pol-encoded integrase (pol IN), and env-encoded gp41 (env gp41) immunodominant region (IDR) sequences were characterized to establish group and subtype designation and to evaluate the degree of genetic diversity at primer and probe binding sites of the viral load assays. Phylogenetic analysis revealed that this virus is an intersubtype B/G recombinant strain. The gag p24 region is subtype G, env gp41 IDR is subtype B, and pol IN is a B/G chimera. Nucleotide mismatches within primer and probe-binding sites provided the molecular basis for differences in quantitation observed between viral load assays. Genetic diversity of HIV-1 continues to challenge the reliability of detection and quantitation by viral load assays.

Serotyping and genotyping of HIV-1 infection in residents of Khayelitsha, Cape Town, South Africa

It is estimated that between 5.5 and 6.1 million people are infected with HIV/acquired immunodeficiency syndrome (AIDS) in South Africa, with subtype C responsible for the majority of these infections. The Khayelitsha suburb of Cape Town has one of the highest HIV prevalence rates in South Africa. Overcrowding combined with unemployment and crime in parts of the area perpetuates high-risk sexual behavior, which increases exposure to infection by HIV. Against this background, the objective of this study was to characterize HIV-1 in residents confirmed to be seropositive. Serotyping was performed through a competitive enzyme-linked immunosorbent assay (cPEIA). Genotyping methods included RNA isolation followed by RT-PCR and sequencing of the gag p24, env gp41 immunodominant region (IDR), and env gp120 V3 genome regions of HIV-1. With the exception of a possible C/D recombinant strain, all HIV-1 strains were characterized as HIV-1 group M subtype C. One individual was shown to harbor multiple strains of HIV-1 subtype C. In Southern Africa, the focus has been to develop a subtype C candidate vaccine, as this is the major subtype found in this geographical area. Therefore, the spread of HIV-1 and its recombinant strains needs to be monitored closely.

Characterization of a Novel Human Immunodeficiency Virus Type 1 Neutralizable Epitope within the Immunodominant Region of gp41

Previously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600–614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits.

Mapping of the interaction between the immunodominant loop of the ectodomain of HIV-1 gp41 and human complement protein C1q.

The human immunodeficiency virus type 1 transmembrane envelope glycoprotein gp41 has been previously shown to activate the C1 complex of human complement through direct interaction with its C1q subunit. The major interaction site has been located within the gp41 immunodominant region (residues 590-620), and a synthetic peptide overlapping residues 601-613 of gp41 (sequence GIWGCSGKLICTT) was shown to inhibit binding of gp41 to C1q in vitro (Thielens, N.M., Bally, I.M., Ebenbichler, C.F., Dierich, M.P. & Arlaud, G.J. (1993) J. Immunol. 151, 6583-6592). The ectodomain of gp41 (s-gp41) was secreted from the methylotrophic yeast Pichia pastoris and purified by immunoaffinity chromatography. Enzymatic deglycosylation of the recombinant s-gp41 was necessary to allow its in vitro interaction with C1q. A solid-phase competition assay was used to monitor the effect of mutant peptides derived from segment 601-613 of gp41 on the binding of deglycosylated s-gp41 to C1q. Whereas mutation of Ser606 had no effect, replacement of Ile602, Trp603, Lys608, Leu609 and Ile610 by Ala abolished the ability of the resulting peptides to inhibit binding of s-gp41 to C1q, suggesting that these residues participate in the interaction between gp41 and C1q. These findings are discussed in the light of a structural model of the immunodominant loop of gp41. It is proposed that the recognition of gp41 by C1q is driven by hydrophobic interactions, and that the sites of gp41 responsible for interaction with gp120 and C1q partly overlap.

Phylogeny of HIV type 1 group O isolates based on env gene sequences.

769 H UM A N IM M UNOD EFIC IEN CY VIRU S TY PE 1 (HIV-1) can be classified in two major groups, M (main) and O (outlier), although the emergence of a new cluster of equidistant isolates, termed group N, has been reported. 1 Whereas group M can be subdivided into 10 different subtypes (A±J), a possible distinction of clades within group O has been limited by the sequence information available in the current database. A subtype A-O has been proposed for a group of 11 HIV-1 group O isolates (ANT70, BCF01, BCF02, BCF03, BCF07, BCF08, BCF13, ESP1, ESP2, ESP3, and ESP4) on the basis of sequences belonging to the reverse transcriptase gene, whereas the other isolates analyzed (MVP5180, VAU, RUD, BCF06, and BCF11) did not form a defined cluster. Since its first description in 1990, HIV-1 group O infection has been reported in West Africa, Europe, and the United States, almost exclusively in native Cameroonians and their contacts. In this sequence note we describe env gene sequences corresponding to the C2V3-coding region of gp120 and to the immunodominant region of gp41. The sequences belong to four HIV-1 group O-infected patients identified in Spain up to November 1998. The sequences and a phylogenetic tree derived from all available sequences of these genomic regions support the existence of a defined subtype, termed A-O, within this highly heterogeneous group of viruses. Isolates ESP1, ESP2, ESP3, and ESP4 were obtained from patients at Hospital Carlos III in Madrid, Spain. ESP1 and ESP2 are from a heterosexual couple diagnosed with HIV-1 infection in 1995. ESP3 and ESP4 are from a 48-year-old man and a vertically infected 14-year-old boy, respectively, who were diagnosed with HIV-1 group O infection in 1997. Their main clinical and epidemiologica l features have been described elsewhere, and are summarized in Table 1. Peripheral blood mononuclear cells (PBMCs) were collected from these individuals using standard protocols. Proviral DNA was obtained from cryopreserved PBMCs (1-10 3 10 cells) by proteinase K digestion, phenol extraction and ethanol precipitation. DNA amplification of the C2V3-coding region of the env gene was carried out using a nested-PCR strategy as previously described. The gp41 immunodom inant coding region was amplified by nested PCR using two sets of primer pairs. The outer primers were gp41A (5 9 -CAACT ACAAA GTAGT AAGGG3 9 ), and gp41B (5 9 -CTACT AGTGC TCCTA CTATG-3 9 ), whose 5 9 -ends correspond to positions 7718 and 8352, respectively, according to the ANT70 isolate (GenBank accession number L20587). The inner primers were gp41C (5 9 -CAGTA GGATT GGGAA TGCTA-3 9 ), and gp41D (5 9 -CCATT TAGTT ATGTC AAGCC-3 9 ), whose 5 9 -ends correspond to positions 7813 and 8306, respectively, according to the ANT70 isolate. Consensus nucleotide sequences between positions 6849 and 7253, and 7814 and 8267 (numbering according to the HIV-1 ANT70 isolate), which correspond to the C2V3-coding and gp41-imm unodom inant regions, respectively, were determined as previously described. 11 Nucleotide sequences were compiled and translated by SEQED and TRANSLATE programs from the GCG package. 13 Multiple sequence alignments were carried out using the CLUSTALW program, 14 and sequence and phylogenetic analyses were performed using the PHYLIP software package version 3.5. Nucleotide sequence data from the C2V3-coding region and the gp41 immunodominant region reported in this sequence note have been deposited in the GenBank nucleotide sequence database (accession numbers AF081811 to AF081817). We have compared the sequences encoding the immunodominant region of gp41 determ ined for isolates ESP1, ESP2, ESP3, and ESP4 with sequences of the corresponding region of other representative HIV-1 group O isolates available in the current database. The comparison included isolates ANT70, MVP5180, VAU, vi686, and DUR (GenBank accession num-

Enhancing antibodies in HIV infection.

The author has summarized the history of discovery, the mechanism and the clinical significance of antibody-dependent enhancement (ADE) of HIV infection. ADE has two major forms: (a) complement-mediated antibody-dependent enhancement (C-ADE) and (b) complement-independent Fc receptor-dependent ADE (FcR-ADE). The most important epitope responsible for the development of C-ADE-mediating antibodies is present in the immunodominant region of gp41 while antibodies mediating FcR-ADE react mainly with V3 loop of gp120. There are at least three fundamentally different hypotheses for the explanation of ADE in vitro: (a) increased adhesion of HIV -antibody-(complement) complexes to FcR or complement receptor carrying cells; (b) facilitation of HIV-target cell fusion by complement fragment deposited on the HIV-virions and (c) complement activation products may have a non-specific stimulatory effect on target cells resulting in enhanced virus production. FcR-ADE and C-ADE have been measured in vitro mostly by using FcR-carrying and complement receptor-carrying cell lines, respectively; no efforts have been made to standardize these methods. Several data support the possible clinical significance of FcR-ADE and C-ADE: (a) Cross-sectional and longitudinal studies indicate a correlation between the amounts of FcR-ADE and C-ADE-mediating antibodies and clinical, immunological and virological progression of the HIV-disease; (b) ADE may facilitate maternal-infant HIV-1 transmission; (c) According to experiments in animal models, ADE are present and may modify the course of SIV (simian immunodeficiency) infection as well. The author raises a new hypothesis on the mechanism of the in vivo effect of C-ADE. According to the hypothesis, C-ADE-mediating antibodies exert their effect through enhancement of HIV propagation and consequent facilitation of the progression of HIV disease. Finally, according to observations from animal experiments and human clinical trials it cannot be excluded that ADE-mediating antibodies may develop, diminish the beneficial effect or may be harmful in volunteers vaccinated with HIV-1 candidate vaccines.

Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry.

The immunologic relatedness of the various human immunodeficiency virus type 1 (HIV-1) clades was determined with 13 human anti-HIV-1 monoclonal antibodies (MAbs) to six immunogenic regions of the HIV-1 structural proteins. The immunoreactivity of the native, oligomeric viral envelope glycoproteins expressed on the surfaces of human peripheral blood mononuclear cells infected in vitro with primary isolates from clades A through E was determined by flow cytometry. Some epitopes in the immunodominant region of gp41 and the C terminus of gp120 appear to be HIV-1 group specific in that they are expressed on the surfaces of cells in cultures infected with the majority of viruses tested from clades A to E. Epitopes within the V3 region appear to be clade restricted. Surprisingly, one MAb to an epitope in the C terminus of gp120 was entirely clade B specific. Staining with anti-V2 and anti-CD4 binding domain (CD4bd) reagents was infrequently detected. Anti-CD4bd MAbs stained only CD4-negative T cells because the CD4bd of gp120 appeared to be complexed with membrane CD4. When present, the epitopes of V2 and the CD4bd appeared to be expressed on cells infected with various clades. Thus, the results suggest that MAbs to gp41, the C terminus, and the V3 loop of gp120 are most useful in serotyping primary isolates of HIV-1, providing group-specific, clade-restricted, and clade-specific reagents. The use of the immunofluorescent method with the reagents described herein distinguishes infection with clade B from that with all other HIV-1 clades. With additional MAbs, this technique will allow a broadly applicable, reproducible, and practical method for serotyping HIV-1.

Molecular mimicry between HIV-1 gp41 and an astrocyte isoform of alpha-actinin.

A 100-kDa astrocyte antigen previously shown to cross-react with a monoclonal antibody (MAb) generated against amino acids (aa) 598 to 609 of the transmembrane protein gp41 of human immunodeficiency virus type 1 [HIV-1] has now been molecularly characterized and found to be an alpha-actinin (alpha-actinin) related protein. Western blot analyses of human astrocytoma cells fractionated by differential centrifugation and detergent phase separation showed that the antigen was membrane associated. The astrocyte protein was purified to apparent homogeneity by immunoaffinity chromatography. Amino acid analysis of three peptide fragments obtained by cleavage of the purified 100-kDa protein revealed sequence identities of 77, 83 and 100% to a non-muscle isoform of human alpha-actinin. In addition, the aa 598-609 sequence of gp41 recognized by MAb 781.4, and the aa 581-597 sequence recognized by another cross-reactive MAb 781.3, were 73% and 53% similar to regions of alpha-actinin. This molecular mimicry between gp41 and alpha-actinin was supported by antibody cross-reactivity in Western immunoblot and ELISA analyses. Both anti-gp41 and anti-alpha-actinin MAbs bind to the surface of the human astrocytoma cells as detected by a cell surface binding assay and immunofluorescence. Antibodies made against this immunodominant region of gp41 in the serum and CSF of HIV-infected individuals have access to astrocytes within the CNS. The identification of the astrocyte antigen as an alpha-actinin related protein will allow further work to determine how this immunological cross-reactivity could perturb astrocyte function and contribute to HIV neuropathology.

Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160

In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens.