High-mannose-specific deglycosylation of HIV-1 gp120 induced by resistance to cyanovirin-N and the impact on antibody neutralization

HIV-1 uses glycans on gp120 to occlude its highly immunogenic epitopes. To better elucidate escape mechanisms of HIV-1 from carbohydrate-binding agents (CBA) and to understand the impact of CBA-escape on viral immune evasion, we generated and examined the biological properties of HIV-1 resistant to cyanovirin-N (CV-N) or cross-resistant to additional CBAs. Genotypic and phenotypic characterization of resistant env clones indicated that 3-5 high-mannose residues from 289 to 448 in the C2-C4 region of gp120 were mutated and correlated with the resistance levels. The specificity and minimal requirements of deglycosylation for CV-N resistance were further assessed by mutagenesis study. The sensitivity of resistant variants to a range of CBAs, immunoglobulins, sera and monoclonal antibodies (MAb) were investigated. For the first time, our data have collectively defined the high-mannose residues on gp120 affecting CV-N activity, and demonstrated that CBA-escape HIV-1 has increased sensitivity to immunoglobulins and sera from HIV patients, and particularly to V3 loop-directed MAbs. Our study provides a proof-of-concept that targeting HIV-1 glycan shields may represent a novel antiviral strategy.     

Detection of orientation-specific anti-gp120 antibodies by a new N-glycanase protection assay

Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans.     

A comparative immunogenicity study of HIV-1 virus-like particles bearing various forms of envelope proteins, particles bearing no envelope and soluble monomeric gp120

To assess the potential of native Envelope glycoprotein (Env) trimers as neutralizing antibody vaccines, we immunized guinea pigs with three types of VLPs and soluble gp120. Particles included "SOS-VLPs" (bearing disulfide-shackled functional trimers), "UNC-VLPs" (bearing uncleaved nonfunctional Env) and "naked VLPs" (bearing no Env). The SOS-VLPs were found to have a density of about 27 native trimers per particle, approximately twice that of live inactivated HIV-1 preparations. As immunogens, UNC- and SOS-VLP rapidly elicited anti-gp120 antibodies focused on the V3 loop and the gp120 coreceptor binding site. Reactivity to the gp41 immunodominant domain was absent in SOS-VLP sera, presumably because gp120-gp41 association is stabilized, effectively covering this epitope. Gp120-immune sera reacted with the receptor binding sites of gp120 and were less focused on the V3 loop. Some Env-VLP sera neutralized primary isolates at modest titers. The measurement of neutralization was found to be affected by the cell lines used. Depending on the assay particulars, non-Env specific antibodies in VLP sera could enhance infection, or nonspecifically neutralize. However, a neutralization assay using TZM-BL cells was essentially clear of these effects. We also describe a native trimer binding assay to confirm neutralization activity in a manner that completely eliminates nonspecific effects. Overall, our data suggests that Env-VLP sera were primarily focused on nonfunctional forms of Env on VLP surfaces, possibly gp120/gp41 monomers and not the trimers. Therefore, to make progress toward a more effective VLP-based vaccine, we will need to find ways to refocus the attention of B cells on native trimers.     

Characteristics of the env genes of HIV type 1 quasispecies in long-term nonprogressors with broadly neutralizing antibodies

Primary isolates of different subtypes of HIV-1 can be neutralized in vitro by the broadly neutralizing antibodies (NAbs) found in the sera of a small number of HIV-1-infected patients. This broad response is most frequent in long-term nonprogressors (LTNPs). We investigated whether the presence of NAbs in the sera of some LTNPs was associated with particular properties of the envelope glycoproteins of the variants found in these patients. Toward that aim, 147 env gene fragments (encoding almost the entire gp120) amplified from the proviral DNA of 5 LTNPs who developed broadly NAbs (NAb+) and of 4 LTNPs who did not develop such broadly NAbs (NAb-) were cloned, sequenced, and compared. We found that the development of broadly NAbs was associated with high viral loads, greater diversity in the gp120 of the viruses infecting these patients, and longer V1 sequences and additional N-gly sites in V1. In addition, a higher proportion of defective clones was found among the env genes of NAb- patients (25% to 93%)-particularly those with lower viral loads and low levels of env diversity-than among those of NAb+ patients (7% to 19%).     

Characterization of a monoclonal antibody to a novel glycan-dependent epitope in the V1/V2 domain of the HIV-1 envelope protein,gp120

Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of additional antibodies to GDEs.     

Selected amino acid changes in HIV-1 subtype-C gp41 are associated with specific gp120(V3) signatures in the regulation of co-receptor usage

The majority of studies have characterized the tropism of HIV-1 subtype-B isolates, but little is known about the determinants of tropism in other subtypes. So, the goal of the present study was to genetically characterize the envelope of viral proteins in terms of co-receptor usage by analyzing 356 full-length env sequences derived from HIV-1 subtype-C infected individuals. The co-receptor usage of V3 sequences was inferred by using the Geno2Pheno and PSSM algorithms, and also analyzed to the "11/25 rule". All reported env sequences were also analyzed with regard to N-linked glycosylation sites, net charge and hydrophilicity, as well as the binomial correlation phi coefficient to assess covariation among gp120(V3) and gp41 signatures and the average linkage hierarchical agglomerative clustering were also performed. Among env sequences present in Los Alamos Database, 255 and 101 sequences predicted as CCR5 and CXCR4 were selected, respectively. The classical V3 signatures at positions 11 and 25, and other specific V3 and gp41 amino acid changes were found statistically associated with different co-receptor usage. Furthermore, several statistically significant associations between V3 and gp41 signatures were also observed. The dendrogram topology showed a cluster associated with CCR5-usage composed by five gp41 mutated positions, A22V, R133M, E136G, N140L, and N166Q that clustered with T2V(V3) and G24T(V3) (bootstrap=1). Conversely, a heterogeneous cluster with CXCR4-usage, involving S11GR(V3), 13-14insIG/LG(V3), P16RQ(V3), Q18KR(V3), F20ILV(V3), D25KRQ(V3), Q32KR(V3) along with A30T(gp41), S107N(gp41), D148E(gp41), A189S(gp41) was identified (bootstrap=0.86). Our results show that as observed for HIV-1 subtype-B, also in subtype-C specific and different gp41 and gp120V3 amino acid changes are associated individually or together with CXCR4 and/or CCR5 usage. These findings strengthen previous observations that determinants of tropism may also reside in the gp41 protein.     

Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4

Summary Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.     

The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4(+) cells

It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4⁺ cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4⁺ cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides.     

HIV-specific lymphoproliferative responses in asymptomatic HIV-infected individuals.

During HIV-1 infection, the current tenet is that only anti-gag antibodies decline, while those directed at env remain stable. Among the latter antibodies, those directed to the V3 domain of gp120 are assumed to play a role in the immune surveillance against HIV-1. We investigated the correlation between anti-V3 antibody levels and the clinical stage of infection and the ability to neutralize syncytium formation. Using a V3-specific antigen-limited ELISA, we analysed the antibody levels of a panel of 93 HIV-1+ sera to V3 peptides derived from different HIV-1 strains and from the North American/European consensus sequence V3(Cs). Sera preferentially recognized V3 peptides from the representative V3(MN) strain and V3(Cs). Antibody reactivity to V3(MN) or V3(Cs) actually declined in relation with progression to AIDS, while antibodies against whole recombinant gp160 or gp41 immunodominant epitope remained stable. There was a strong correlation (P < 0.0001) between anti-V3 (Cs)/V3(MN) antibody levels and serum titres that neutralized HIV-1MN-mediated syncytia. Serology based on V3-specific antigen-limited ELISA indicates that anti-V3 antibody reactivity may decline during the course of HIV infection.     

The N-linked glycan g15 within the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage,cellular tropism,and neutralization

We have studied infectivity and neutralization of X4, R5, and R5X4 tropic HIV-1 mutants, which are lacking N-linked glycosylation sites for glycans g13, g14, g15, and g17 in the V3 loop region of gp120. X4-tropic NL4-3 mutants lacking combinations of g14/15 or g15/17 showed markedly higher infectivity in CXCR4-specific infection. The role of g15 in CCR5-specific infection was investigated using viruses with high (NL-918, R5-monotropic), medium (NL-991, R5-monotropic), and low (NL-952, R5X4-dualtropic) CCR5-specific infectivity. For NL-991, a reduction of infectivity on GHOST-CCR5 cells was observed for a mutant lacking g15. For NL-952 mutants all lacking g15, a complete loss of CCR5-specificity was observed and NL-952 was shifted from R5X4 to X4 tropism. For all mutants of NL4-3, NL-991, and NL-952, where the lack of g15 markedly influenced infectivity or coreceptor usage, neutralization was enhanced. In contrast, NL-918 mutants with or without g15 showed no difference in neutralization and no difference in GHOST-CCR5 infection rates. Thus, for viruses with a low or medium CCR5-specificity the role of g15 for changing CCR5-usage and sensitivity to neutralization was more significant than for viruses with high infection rates on GHOST-CCR5 cells. Our data demonstrate that V3 glycans play an important role in the usage of CXCR4 and CCR5. The lack of g15 was relevant for a more efficient use of CXCR4, whereas interaction with CCR5 was facilitated in the presence of g15. This study also demonstrates that glycan g15 is involved in blocking of neutralizing antibodies and shifting HIV tropism from R5X4 to X4.     

Evaluation of viral-lysate enzyme-linked immunosorbent assay kits for detecting human immunodeficiency virus (type 1) infections using human sera standardized by quantitative western blotting.

The first generation of proprietary reagents for detecting antibodies to the Human Immunodeficiency Virus Type 1 (HIV-1) by enzyme-linked immunosorbent assay (ELISA) used as antigen partially purified virus from cell culture lysates. These tests, which are still in use, may vary in their antibody measurement capabilities if different proportions of the viral polypeptides are present in the viral lysate mixtures. We determined the quantities of antibodies in the serum of persons infected with HIV-1 by dilution analysis using 3 ELISA kits: Abbott [A], Du Pont [D], Genetic Systems [G]. The proportionate antibody titres of each serum to p24gag and gp160env/120env were established by quantitative Western blotting. Serum antibody titres were high, frequently over 1:10,000, a result observed both by ELISA and Western blot. For Kit D, sera with high proportions of antibody to p24gag produced antibody titration curves with steep slopes whereas shallower slopes were found in sera with high proportions of antibody to gp160env. In contrast, Kit A gave steeper slopes with sera enriched for gp160env antibodies. Kit G gave results with slopes intermediate between Kits A and D. Serum antibody titres differed between kits depending upon the proportion and concentration of antibodies in a given serum to gp160env and p24gag. The findings that both the concentration and proportion of antibodies to specific viral polypeptides in human sera markedly affect the signal intensity produced by proprietary ELISAs suggest the need for several control sera which reflect the diversity of human serum responses. Standardization of human reference sera by quantitative Western blotting will assist in evaluation and quality control of ELISA tests.     

Structure-based,targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.     

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.     

Highly conserved HIV-1 gp120 glycans proximal to CD4-binding region affect viral infectivity and neutralizing antibody induction

Glycosylation plays important roles in gp120 structure and HIV-1 immune evasion. In the current study, we introduced deglycosylations into the 24 N-linked glycosylation sites of a R5 env MWS2 cloned from semen and systematically analyzed the impact on infectivity, antigenicity, immunogenicity and sensitivity to entry inhibitors. We found that mutants N156-T158A, N197-S199A, N262-S264A and N410-T412A conferred decreased infectivity and enhanced sensitivity to a series of antibodies and entry inhibitors. When mice were immunized with the DNA of wild-type or mutated gp160, gp140 or gp120; N156-T158A, N262-S264A and N410-T412A were more effective in inducing neutralizing activity against wild-type MWS2 as well as heterologous IIIB and CH811 Envs. In general, gp160 and gp140 induced higher neutralizing activity compared with gp120. Our study demonstrates for the first time that removal of individual glycan N156, N262 or N410 proximal to CD4-binding region impairs viral infectivity and results in enhanced capability to induce neutralizing activity.     

Effects of Mutations in Constant Regions 3 and 4 of Envelope of Simian Immunodeficiency Virus

Twenty-six mutant forms of simian immunodeficiency virus strain mac239 were constructed with changes in constant region 4 (C4) of env. Twenty-four of these had a single amino acid change, one had changes in two amino acids, and one had a deletion of eight amino acids. The effects of these mutations on viral replication, gp160 processing, and binding of env protein to soluble CD4 receptor were analyzed. The C4 region was relatively sensitive to sequence changes since only 11 of the 26 mutants replicated appreciably. Eight of the 15 mutants that were replication incompetent exhibited grossly defective processing of the gp160 env precursor; these mutations likely resulted in global effects on gp160 structure. Six of the replication incompetent mutants exhibited normal or near normal gp160 processing and binding of any protein to sCD4 and thus were probably blocked at some step subsequent to binding of virus to its CD4 receptor Only one of the C4 mutations, 441W → R, resulted in greatly decreased binding to sCD4 while retaining normal processing of gp160. The equivalent residue in HIV-1 has similarly been shown previously to be important for binding of HIV-1 to the CD4 receptor. Since a W → S mutation at position 441 in C4 of SIVmac239 affected both gp160 processing and sCD4 binding, it is not clear whether the 441 tryptophan is actually important for contacting CD4 or for maintaining an appropriate configuration. Mutations within a highly conserved GGDPE sequence in C3 of SIVmac239 specifically affected CD4 binding, which is also similar to previous findings with HIV-1. These results demonstrate similar sequence requirements in SIVmac and HIV-1 env for binding CD4, but they raise doubts as to whether C4 sequences are directly involved in the binding.     

Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop

Summary It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation of such threonine or serine residues could decrease the sensitivity to infectivity inhibition by Tn or sialosyl-Tn specific antibodies. All potentially O-glycosylated threonine and serine residues in the V3 loop of cloned HIV-1_BRU were mutagenized to alanine thus abrogating any O-glycosylation at these sites. Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp120.     

Induction of HIV-1 envelope (gp120)-specific cytotoxic T lymphocyte responses in mice by recombinant CHO cell-derived gp120 is enhanced by enzymatic removal of N-linked glycans

Priming of CD8⁺ cytotoxic T lymphocyte (CTL) responses with recombinant proteins has been facilitated by the development of novel adjuvants that deliver antigens into the class I major histocompatibility complex (MHC) pathway. However, the extent to which secondary structure or glycosylation of these proteins prevents priming of class I MHC-restricted CTL responses is not clear. To address this issue, recombinant HIV-1 gp120 envelope proteins produced in yeast, insect, or mammalian cells were compared for the ability to elicit CD8⁺ CTL activity in mice. Envelope-specific CD8⁺ T lymphocytes were detected in BALB/c mice immunized with env 2-3, a 55-kDa yeast-derived envelope protein that is not glycosylated and lacks a native conformation. This response was directed against a previously described epitope in the V3 region of gp120, as well as a newly identified epitope located near the carboxy-terminus of the molecule. Similar levels of V3-directed CTL activity were observed in mice immunized with recombinant gp120 produced in insect (Spodoptera fugiperda) cells using a baculovirus expression system (gp120BAC). In contrast, induction of CTL responses was considerably less efficient when mice were immunized with gp120CHO, a native, fully glycosylated envelope protein produced in mammalian CHO cells. Denaturation of gp120CHO prior to immunization was not sufficient to prime CTL responses. However, envelope-specific CD8⁺ CTL activity was elicited when N-linked glycans were removed by treatment with an endoglycosidase. Possible mechanisms by which N-linked glycans influence delivery or processing of recombinant proteins for class I MHC presentation, and the implications of these findings for the design of subunit vaccines, are discussed.     

Serological detection of infection with diverse human and simian immunodeficiency viruses using consensus env peptides

Cross-species transmission has been shown to play an important role in the emergence of human retroviruses. We developed a generic enzyme immunoassay using synthetic peptides from gp41 and C2V3 consensus sequences (human immunodeficiency virus [HIV] type 1 [HIV-1] groups M, O, and N and the homologous region of simian immunodeficiency virus [SIV] strains from chimpanzees [SIVcpz], SIVcpzGAB1 and SIVcpzANT) to detect divergent HIV and SIV. A cocktail of peptides from gp41 and C2V3 (M-O) detected all HIV-1 group M and O sera and showed cross-reactivity with SIVcpz sera. Further, a mixture of C2V3 peptides (GAB1-ANT) failed to detect HIV-1 infections but reacted with all SIVcpz sera, allowing discrimination of SIVcpz from HIV-1 infections. Since most SIVcpz sera cross-reacted with HIV-1 peptides, we next evaluated SIVcpz serum reactivity with rapid tests for HIV-1/2. SIVcpzANT and SIVcpzUS sera reacted with the Sero-strip and Multispot assays. Both tests are sensitive in detecting group M (97 100%, respectively), although Multispot has lower sensitivity for group O detection (67%) than does Sero-strip (100%). The limited volume and time required to perform these assays make them a generic tool for field screening. The env peptide-based assay and rapid tests should allow for the identification of emerging variants of HIV and SIV.     

Immunization with HIV-1 SF162-derived Envelope gp140 proteins does not protect macaques from heterologous simian-human immunodeficiency virus SHIV89.6P infection

Immunization by the SF162gp140 or the DeltaV2gp140 HIV-1 envelope proteins results in the generation of strong homologous neutralizing antibodies (NAbs) that offer similar degree of protection from disease-development to macaques challenged with homologous virus. These two immunogens elicit weak cross-reactive NAbs and their effectiveness against heterologous challenge is currently unknown. To examine this issue, we immunized macaques with SIVGag p55 and either the SF162gp140 or the DeltaV2gp140 and challenged them intravenously with SHIV-89.6P. All animals became infected but previous immunization with SF162gp140 accelerated the development of anti-SHIV89.6P neutralizing antibody responses following infection. DeltaV2gp140 is derived from SF162gp140 following the deletion of 30 amino acids and one N-linked glycosylation site from the V2 loop. Our results suggest that even small differences in HIV Envelope immunogen structure can affect the neutralizing antibody responses generated following infection.     

A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2

This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1env, incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu³⁺ chelate or with nanoparticles doped with the Eu³⁺ chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu³⁺ chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios.