Development and evaluation of an HIV-1 transfection-neutralization assay.

We developed a transfection-neutralization assay for human immunodeficiency virus type 1 (HIV-1) infectious molecular clones. In this assay CD4 negative adherent cells, transfected in microtiter plates with fixed amounts of proviral DNA of molecular HIV-1 clones, are cocultivated with CD4 positive T cell lines or primary peripheral blood mononuclear cells (PBMC) in the presence of anti-HIV-1 sera or monoclonal antibodies (MAbs). Results obtained with this technique were reproducible and compared favorably with a conventional cell-free infection inhibition assay. The transfection-neutralization assay obviates the need for virus stock preparation and, therefore, is particularly suitable for the evaluation of HIV-1 clones with slow replication kinetics and of recombinant chimeric HIV-1 clones inclined to undergo additional mutations during stock preparation. The potential value of this assay for the analysis of the specificity of neutralizing sera and MAbs was demonstrated in experiments with V3 chimeric molecular clones.

Frequent and early HIV-1MN neutralizing capacity in sera from Dutch HIV-1 seroconverters is related to antibody reactivity to peptides from the gp120 V3 domain.

The temporal development of HIV-1 neutralizing activity and antibodies to the gp120-V3 neutralization domain were studied in sera from 20 Dutch HIV-1-infected individuals followed from seroconversion on. Serum neutralizing capacity was assessed with three T cell line-tropic isolates: HIV-1MN, HIV-1HXB2, and the patient isolate HIV-1(320). Neutralizing activity to HIV-1MN developed in 18 individuals (90%) within 0 to 10 months after seroconversion. Parallel evolution of IgG reactivity to V3 peptides of United States/European type variants, and the capability of such peptides to completely inhibit HIV-1MN neutralization in four of five tested sera (taken 1-2 years after seroconversion), indicate that a large proportion of HIV-1MN neutralizing antibodies is directed to V3. The early appearance and high frequency of HIV-1MN neutralizing activity in the Dutch study group indicate the close relationship of HIV-1MN to HIV-1 variants circulating in the Netherlands. Neutralizing activity to HIV-1HXB2 (in 15 of 20 individuals) developed several months after that to HIV-1MN in all individuals (average, 10 months after seroconversion) and was not seen in the absence of HIV-1MN neutralizing activity. Neutralizing activity to the Dutch isolate HIV-1(320) (found in 11 of 18 tested individuals) emerged simultaneously with that to HIV-1MN in 4 individuals but appeared later in 7. In most individuals, HIV-1HXB2 neutralization was not accompanied by reactivity to a V3 peptide from this strain, indicating that the extension of neutralizing activity to more divergent strains, which takes place at later stages, must be attributed to non-V3-directed antibodies.

Structural and functional analysis of negatively charged milk proteins with anti-HIV activity.

Several polyanionic reagents such as dextran sulfates, heparin sulfates, and negatively charged proteins have been reported to exhibit anti-HIV activity in vitro. Particularly potent inhibition has been reported for the milk protein beta-lactoglobulin (betaLG) on modification by 3-hydroxyphthalic anhydride (3HP). The introduction of multiple negatively charged carboxyl groups along the polypeptide backbone obviously leads to repulsion within the protein molecule and this is likely to affect the specific tertiary, and perhaps also secondary, structure of the protein. We used several biophysical techniques to probe the structural changes that occur on 3HP modification of betaLG. The results suggest that the protein becomes largely unstructured on chemical modification. Although a profound anti-HIV activity was measured for 3HP-betaLG, similar antiviral effects were observed with two other 3HP-modified milk proteins, alpha-lactalbumin and alpha(S2)-casein, but not with the unmodified proteins. Most potent inhibition of HIV-1 replication was obtained with 3HP-modified alpha-lactalbumin, which also demonstrated the least cytotoxicity. These combined results indicate that HIV inhibition is a general property of negatively charged polypeptides and do not support a model in which the negatively charged 3HP-betaLG protein interacts in a structure-specific manner with the CD4 cell surface receptor for HIV-1 entry.

Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.

Limiting deoxynucleoside triphosphate concentrations emphasize the processivity defect of lamivudine-resistant variants of human immunodeficiency virus type 1 reverse transcriptase.

The nucleoside drug lamivudine (3TC) triggers the selection of resistant forms of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with a substitution of amino acid 184Met. The 3TC-resistant RT enzymes 184Val and 184Ile exhibit a processivity defect in in vitro assays that correlates with reduced replication of the corresponding virus variants in primary cells. However, no replication defect is apparent for these two mutants in the transformed T-cell line SupT1. One obvious difference between the two cell types is the intracellular deoxynucleoside triphosphate (dNTP) level. Primary cells have a much smaller dNTP pool, and this cellular condition may emphasize the processivity defect of the codon 184 RT variants. Alternatively, cell-specific cofactors that influence the process of reverse transcription may exist. Such accessory factors may be packaged into the virion to exert an effect on the RT enzyme. To discriminate between these possibilities we performed additional assays with the wild-type and mutant RT enzymes. The RT proteins were either isolated from virions produced by primary and transformed cell types or expressed as recombinant protein. We also performed infection assays with cells treated with a drug that reduces the intracellular dNTP pool. Furthermore, reverse transcription was studied within virus particles in the endogenous assay, which allows for the manipulation of the dNTP level. The combined results indicate that the enzymatic defect of the 3TC-resistant HIV-1 variants is stressed at low dNTP concentrations.

Genomic diversity and antigenic variation of HIV-1: links between pathogenesis, epidemiology and vaccine development.

Recent analysis of primate lentivirus genomes indicates that lentiviruses have infected primates for hundreds of years. The pathogenicity of such viruses may fluctuate due to the high evolution rate of some parts of the viral genome. Fixed nucleic acid substitutions in the gag gene appear to be caused by random fixation of selectively neutral mutants, whereas nonrandom fixation of selectively advantageous mutants, as has been observed for MHC molecules and serine protease inhibitors, appears to be operational for some hypervariable env gene regions. The former is characterized by an excess of silent mutations independent of the rate of change, the latter by an excess of nonsilent mutations. This latter type of selection may especially characterize the third variable region of the external HIV envelope (V3), which contains the principal neutralization domain.

Increased polymerase fidelity of the 3TC-resistant variants of HIV-1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These 3TC-resistant RT variants have a single point mutation that changes the 184Met residue into either Val or Ile. Both codon 184 variants are frequently observed in 3TC-treated patients and can also be selected in cell culture infections. We demonstrated previously that the 184Ile and 184Val RT enzymes exhibit a processivity defect in in vitro assays, with 184Ile being the least processive enzyme [Met(wt) >Val >Ile]. In this study, we measured the polymerase fidelity of the wild-type (184Met) and 3TC-resistant RT enzymes (184Ile and 184Val) on DNA and RNA templates. Both virion- extracted and Escherichia coli -expressed recombinant RT enzymes were used to measure the nucleotide misinsertion and mispair extension efficiencies. The 3TC-resistant enzymes were more accurate than the wild-type RT protein in both type of assays. The order of accuracy observed for the codon 184 variants [Ile >Val >Met(wt)] may suggest an inverse correlation between the fidelity and processivity properties of these enzymes.

Neutralizing activity of anti-peptide antibodies against the principal neutralization domain of human immunodeficiency virus type 1.

Monoclonal antibodies (MAbs) raised against a 15-mer peptide representing the centre of the principal neutralization domain of human immunodeficiency virus type 1 (strain BH10) showed wide variations in neutralizing activity against the homologous strain. The nature of this difference in neutralizing activity was studied by measuring antibody concentration, their affinity for peptide and specificity, by reaction with peptides which differed in the extent of sequence overlap, length and the presence of single amino acid replacements. All MAbs bound to approximately the same region in the principal neutralization domain, within the sequence RIQRGPGRAFV. The peptides with which each antibody was able to react differed by only a few amino acids. The neutralizing activity of each MAb preparation was related to its affinity and concentration; the affinity is related in part to the fine structure of the epitope recognized. MAbs with high affinity for the peptide tended to react only with peptides in which amino acid replacements did not affect the beta-turn potential of the peptide, whereas the reactivity of MABs with low affinity was relatively insensitive to amino acid replacements affecting the beta-turn potential.

Mutations in human immunodeficiency virus type 1 gp41 affect sensitivity to neutralization by gp120 antibodies.

Three closely related molecular human immunodeficiency virus type 1 (HIV-1) clones, with differential neutralization phenotypes, were generated by cloning of an NcoI-BamHI envelope (env) gene fragment (HXB2R nucleotide positions 5221 to 8021) into the full-length HXB2 molecular clone of HIV-1 IIIB. These env gene fragments, containing the complete gp120 coding region and a major part of gp41, were obtained from three different biological clones derived from a chimpanzee-passaged HIV-1 IIIB isolate. Two of the viruses thus obtained (4.4 and 5.1) were strongly resistant to neutralization by infection-induced chimpanzee and human polyclonal antibodies and by HIV-1 IIIB V3-specific monoclonal antibodies and weakly resistant to soluble CD4 and a CD4-binding-site-specific monoclonal antibody. The third virus (6.8) was sensitive to neutralization by the same reagents. The V3 coding sequence and the gp120 amino acid residues important for the discontinuous neutralization epitope overlapping the CD4-binding site were completely conserved among the clones. However, the neutralization-resistant clones 4.4 and 5.1 differed from neutralization-sensitive clone 6.8 by two mutations in gp41. Exchange experiments confirmed that the 3' end of clone 6.8 (nucleotides 6806 to 8021; amino acids 346 to 752) conferred a neutralization-sensitive phenotype to both of the neutralization-resistant clones 4.4 and 5.1. From our study, we conclude that mutations in the extracellular portion of gp41 may affect neutralization sensitivity to gp120 antibodies.

Genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains.

Live, attenuated viruses have been the most successful vaccines in monkey models of human immunodeficiency virus type 1 (HIV-1) infection. However, there are several safety concerns about using such an anti-HIV vaccine in humans, including reversion of the vaccine strain to virulence and recombination with endogenous retroviral sequences to produce new infectious and potentially pathogenic viruses. Because testing in humans would inevitably carry a substantial risk, we set out to test the genetic stability of multiply deleted HIV constructs in perpetuated tissue culture infections. The Delta3 candidate vaccine strain of HIV-1 contains deletions in the viral long terminal repeat (LTR) promoter and the vpr and nef genes. This virus replicates with delayed kinetics, but a profound enhancement of virus replication was observed after approximately 2 months of culturing. Analysis of the revertant viral genome indicated that the three introduced deletions were maintained but a 39-nucleotide sequence was inserted in the LTR promoter region. This insert was formed by duplication of the region encoding three binding sites for the Sp1 transcription factor. The duplicated Sp1 region was demonstrated to increase the LTR promoter activity, and a concomitant increase in the virus replication rate was measured. In fact, duplication of the Sp1 sites increased the fitness of the Delta3 virus (Vpr/Nef/U3) to levels higher than that of the singly deleted DeltaVpr virus. These results indicate that deleted HIV-1 vaccine strains can evolve into fast-replicating variants by multiplication of remaining sequence motifs, and their safety is therefore not guaranteed. This insight may guide future efforts to develop more stable anti-HIV vaccines.

Association of antibodies blocking HIV-1 gp 160-sCD4 attachment with virus neutralizing activity in human sera.

Sera, from HIV-1 and HIV-2 seropositive individuals, were tested for the presence of antibodies able to inhibit the binding (BI) of HIV-IIIB gp 160 (produced in mammalian cells using a vaccinia expression system) to the extracellular portion of the CD4 receptor. A competition enzyme immunoassay (EIA) with soluble CD4 (sCD4) was used. BI antibodies were highly prevalent among HIV-1 seropositives but not in HIV-2 infected individuals. Attempts to localize the binding site for these BI antibodies on the primary sequence of gp 120 by using synthetic peptides encompassing the putative CD4 binding site on gp 120 (aa 397-439) were not successful. This study did not reveal a significant correlation between the presence of BI antibodies and disease evolution. BI antibody titres correlated less well with anti-gp 160 titres (r = 0.51, P less than or equal to 0.011) than with neutralizing antibody (NA) titres using either the isolates HIV-SF2 (SF2) (r = 0.77, P less than or equal to 0.000) and HIV-MN (MN) (r = 0.61, P less than or equal to 0.002) or the isolate HIV-IIIB (HX10) (r = 0.89, P less than or equal to 0.000) of which the gp 160 for the assays was derived. An HIV-IIIB neutralizing serum, elicited in a rabbit by immunization with a 17-mer synthetic peptide derived from the third variable domain (V3) of gp 120, did bind gp 160 without inhibiting the subsequent attachment of sCD4 to gp 160.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial appearance of the 184Ile variant in lamivudine-treated patients is caused by the mutational bias of human immunodeficiency virus type 1 reverse transcriptase.

Treatment of human immunodeficiency virus type 1-infected patients with lamivudine (3TC) results in the appearance of drug-resistant virus variants with a mutation at the 184Met codon (ATG) of the reverse transcriptase (RT) gene. The 184Ile (ATA) variant appears first, but subsequently the 184Val (GTG) variant outcompetes the 184Ile variant. We demonstrated previously that the 184Val enzyme and the corresponding virus are more fit than 184Ile, thereby explaining eventual outgrowth of 184Val. In this study, we set out to determine why 184Ile is usually observed first after initiation of 3TC therapy. With a limiting dilution approach during in vitro selection with 3TC, we measured a significantly higher frequency of the G-->A substitution toward the ATA codon (184Ile; 56%) than the A-->G substitution toward GTG (184Val; 12.5%). This result indicates that the initial appearance of the 184Ile variant in patients is a consequence of the mutation bias of the RT enzyme. Interestingly, a novel 3TC-resistant variant which was generated by T-->C substitution (184Thr; 28%) was also observed. The RT enzyme of the 184Thr variant was less than 10% active compared with the wild-type enzyme, and the replication capacity of this variant was severely reduced. Selection of the 184Thr variant illustrates that the limiting dilution approach allows the selection of drug-resistant variants with suboptimal fitness.

An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization.

Carbohydrate side chains of envelope glycoproteins of HIV-1 and other viruses have been postulated to interfere with binding of neutralizing antibodies. So far, however, little evidence for interference of specific N-glycans with virus neutralization has been provided. We used four infectious HIV-1 molecular clones chimeric for their gp 120 V3 domains to study the influence on HIV-1 neutralization of an N-glycan localized within the V3 loop. Two clones lacking the 301N-glycan were at least 8-fold more sensitive to neutralization by two V3-specific monoclonal antibodies (MAbs) and 2- to 10-fold more sensitive to neutralization by a CD4-binding-site-specific human MAb than two HIV-1 clones glycosylated at this site. The affinity of the V3 MAbs for soluble gp120 of the four clones was similar. However, a decreased binding of these MAbs to the gp120 of the two 301N-glycosylated clones was observed when the majority of gp120 was virion-associated during the initial binding step. These findings indicate that the 301N-glycan may interfere with the binding of neutralizing antibodies by limiting the accessibility of neutralization sites or by inducing conformational changes in the HIV-1 gp120 molecule.

Antibody reactivity to deletion mutants of the HIV-1 SF2 envelope.

In human immunodeficiency virus type 1 (HIV-1) infected individuals, the antibody response to the external envelope (gp120) is associated with in vitro neutralization. To further characterize the anti-gp120 response, we examined the IgG reactivity of 75 HIV-1-seroconverted and 200 HIV-1-seropositive individuals to deletion mutants of gp120 in an enzyme immunoassay. We used yeast-derived, non-glycosylated recombinant HIV-1 SF2 gp120 equivalent and-variants deleted in variable regions. We observed two distinctive response patterns: IgG non-responders (SF2-V3-restricted responders) and IgG responders to conserved regions of gp120. This divergence in response pattern occurred soon after gag/env HIV-1 antibody seroconversion and persisted in time within an individual. In addition, the SF2-V3-restricted responders had a higher frequency of HIV-1 core antigen positivity and HIV-1 core antibody negativity than the non-restricted responders. These results suggest that specific and persistent host antibody response patterns to gp120 develop early in HIV-1 infection and that these patterns are associated with differences in HIV-1 expression.

Comparison and fine mapping of both high and low neutralizing monoclonal antibodies against the principal neutralization domain of HIV-1.

Monoclonal antibodies raised against viral lysate of HIV-1 (strain LAV-1) and against recombinant gp 160 of HIV-1 (strain HTLV IIIB) which neutralized HIV-1 in a type specific manner were mapped with the aid of peptides (Pepscan analysis). Each of these monoclonal antibodies bound to peptides located on the principal neutralizing domain (PND) of HIV-1. We found that the antigenic sites of the MAbs described in this paper are represented by linear peptides of at least 10 amino acids long. The affinity of the MAbs is high for these peptides and in the same order of magnitude as for native gp 160. The fine mapping of the epitopes may reflect structural features of the PND, for instance which amino acid side chains are exposed and which are buried in the protein. Furthermore the fine mapping of the epitopes explained the HIV type-specific neutralizing activity of the MAbs. Antibodies that bound to the tip of the loop (amino acids QRGPGRAF) have a higher neutralizing activity than antibodies that bound to amino acids towards the N-terminal side of the loop (amino acids KSIRI). Furthermore, MAbs that bound to virtually the same amino acids on the tip of the loop (amino acids IQRGPGRAF and RGPGRAFV) had different neutralizing activities due to different affinities for native gp 160. These data reveal that neutralizing activity not only is determined by the affinity of an antibody to the neutralizing site but also by its fine binding specificities to the V 3 loop of gp 120.

Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay.

A simple approach for the determination of drug susceptibilities by using human immunodeficiency virus type 1 (HIV-1) RNA from the sera of patients is described. HIV-1 RNA was extracted from patient sera, and the 5' part of the reverse transcriptase (RT) gene was transcribed into DNA and amplified in a nested PCR. The amplified fragment covers the 3' part of the protease gene and amino acids 1 to 304 of the RT gene. This fragment can be introduced through homologous recombination, as described previously, into a novel HIV-1 reference strain (pHXB2 delta 2-261RT) from which amino acids 2 to 261 of RT have been deleted. The resulting recombinant virus expresses all properties of the HXB2 reference strain except for those encoded by the introduced part of the patient RT gene. Recombinant viruses were subsequently tested for drug susceptibility in a microtiter format killing assay [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay] as well as in the standard HeLa CD4+ plaque reduction assay. Similar susceptibility profiles were obtained by each assay with recombinant viruses derived from patients receiving alternating nevirapine and zidovudine treatment or lamivudine-zidovudine combination therapy. In conclusion, this approach enables high-through-put determination of the drug susceptibilities of serum RNA-derived RT genes, independent of the patient's viral background, and generates the possibility of relating changes in susceptibility to changes in viral genotypes.

Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild-type and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild-type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC-resistant RT mutants produce significantly shorter cDNA molecules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3TC-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC-treated patients that develop drug-resistant HIV-1 variants, this may be of considerable clinical benefit.