Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.     

Impact of V2 mutations on escape from a potent neutralizing anti-V3 monoclonal antibody during in vitro selection of a primary human immunodeficiency virus type 1 isolate

KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.     

Increased neutralization sensitivity of CD4-independent human immunodeficiency virus variants

Naturally occurring human immunodeficiency virus (HIV-1) variants require the presence of CD4 and specific chemokine receptors to enter a cell. In the laboratory, HIV-1 variants that are capable of bypassing CD4 and utilizing only the CCR5 chemokine receptor for virus entry have been generated. Here we report that these CD4-independent viruses are significantly more sensitive to neutralization by soluble CD4 and a variety of antibodies. The same amino acid changes in the HIV-1 gp120 envelope glycoprotein determined CD4 independence and neutralization sensitivity. The CD4-independent envelope glycoproteins exhibited higher affinity for antibodies against CD4-induced gp120 epitopes but not other neutralizing ligands. The CD4-independent envelope glycoproteins did not exhibit increased lability relative to the wild-type envelope glycoproteins. The utilization of two receptors apparently allows HIV-1 to maintain a more neutralization-resistant state prior to engaging CD4 on the target cell, explaining the rarity of CD4 independence in wild-type HIV-1.     

Longer V1V2 region with increased number of potential N-linked glycosylation sites in the HIV-1 envelope glycoprotein protects against HIV-specific neutralizing antibodies

Human immunodeficiency virus type 1 (HIV-1) has the ability to adapt to the host environment by escaping from host immune responses. We previously observed that escape from humoral immunity, both at the individual and at a population level, coincided with longer variable loops and an increased number of potential N-linked glycosylation sites (PNGS) in the viral envelope glycoprotein (Env) and, in particular, in variable regions 1 and 2 (V1V2). Here, we provide several lines of evidence for the role of V1V2 in the resistance of HIV-1 to neutralizing antibodies. First, we determined that the increasing neutralization resistance of a reference panel of tier-categorized neutralization-sensitive and -resistant HIV-1 variants coincided with a longer V1V2 loop containing more PNGS. Second, an exchange of the different variable regions of Env from a neutralization-sensitive HIV-1 variant into a neutralization-resistant escape variant from the same individual revealed that the V1V2 loop is a strong determinant for sensitivity to autologous-serum neutralization. Third, exchange of the V1V2 loop of neutralization-sensitive HIV-1 variants from historical seroconverters with the V1V2 loop of neutralization-resistant HIV-1 variants from contemporary seroconverters decreased the neutralization sensitivity to CD4-binding site-directed antibodies. Overall, we demonstrate that an increase in the length of the V1V2 loop and/or the number of PNGS in that same region of the HIV-1 envelope glycoprotein is directly involved in the protection of HIV-1 against HIV-specific neutralizing antibodies, possibly by shielding underlying epitopes in the envelope glycoprotein from antibody recognition.     

Neutralizing Antibody Escape during HIV-1 Mother-to-Child Transmission Involves Conformational Masking of Distal Epitopes in Envelope

HIV-1 variants transmitted to infants are often resistant to maternal neutralizing antibodies (NAbs), suggesting that they have escaped maternal NAb pressure. To define the molecular basis of NAb escape that contributes to selection of transmitted variants, we analyzed 5 viruses from 2 mother-to-child transmission pairs, in which the infant virus, but not the maternal virus, was resistant to neutralization by maternal plasma near transmission. We generated chimeric viruses between maternal and infant envelope clones obtained near transmission and examined neutralization by maternal plasma. The molecular determinants of NAb escape were distinct, even when comparing two maternal variants to the transmitted infant virus within one pair, in which insertions in V4 of gp120 and substitutions in HR2 of gp41 conferred neutralization resistance. In another pair, deletions and substitutions in V1 to V3 conferred resistance, but neither V1/V2 nor V3 alone was sufficient. Although the sequence determinants of escape were distinct, all of them involved modifications of potential N-linked glycosylation sites. None of the regions that mediated escape were major linear targets of maternal NAbs because corresponding peptides failed to compete for neutralization. Instead, these regions disrupted multiple distal epitopes targeted by HIV-1-specific monoclonal antibodies, suggesting that escape from maternal NAbs occurred through conformational masking of distal epitopes. This strategy likely allows HIV-1 to utilize relatively limited changes in the envelope to preserve the ability to infect a new host while simultaneously evading multiple NAb specificities present in maternal plasma.     

The N-terminal V3 loop glycan modulates the interaction of clade A and B human immunodeficiency virus type 1 envelopes with CD4 and chemokine receptors

We investigated the underlying mechanism by which the highly conserved N-terminal V3 loop glycan of gp120 conferred resistance to neutralization of human immunodeficiency virus type 1 (HIV-1). We find that the presence or absence of this V3 glycan on clade A and B viruses accorded various degrees of susceptibility to neutralization by antibodies to the CD4 binding site, CD4-induced epitopes, and chemokine receptors. Our data suggest that this carbohydrate moiety on gp120 blocks access to the binding site for CD4 and modulates the chemokine receptor binding site of phenotypically diverse clade A and clade B isolates. Its presence also contributes to the masking of CD4-induced epitopes on clade B envelopes. These findings reveal a common mechanism by which diverse HIV-1 isolates escape immune recognition. Furthermore, the observation that conserved functional epitopes of HIV-1 are more exposed on V3 glycan-deficient envelope glycoproteins provides a basis for exploring the use of these envelopes as vaccine components.     

A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.     

Characterization of mutants of human immunodeficiency virus type 1 that have escaped neutralization by a monoclonal antibody to the gp120 V2 loop.

The biologically cloned human immunodeficiency virus type 1 (HIV-1) RF isolate is sensitive to neutralization by the murine monoclonal antibody (MAb) G3-4 to a conformationally sensitive epitope in the V2 loop of HIV-1 gp120. To assess how variation in the V2 amino acid sequence affects neutralization by this MAb, we cultured RF in the presence of G3-4 to select neutralization escape mutants. Three such mutants resistant to G3-4 neutralization were generated from three independent experiments. Solubilized gp120 from each of these escape mutants had a reduced affinity for G3-4 and also for two other V2 MAbs that were able to bind the wild-type RF gp120. PCR sequencing of the entire gp120 of the wild-type RF virus and the escape mutants showed that amino acid substitutions had occurred only at two positions, Y177H and L179P, both in V2. Experimental introduction of the Y177H substitution into the RF V2 loop in the context of the NL4-3 molecular clone re-created the G3-4-resistant phenotype. The L179P mutant was not viable. Thus, our findings confirm that the HIV-1 V2 loop contains the conformationally sensitive neutralization epitope recognized by G3-4 and that a single amino acid substitution within this region can result in escape variants that arise from immune selection pressure.     

Immunologic escape as a mechanism of viral persistence in HIV-1 infection

Two specific immune responses to HIV, the cytolytic T cell response to epitopes in the core/envelope proteins and the antibody neutralization response to the V3 epitope in the envelope, are reviewed. Substantial data has accumulated indicating that virus variants can be isolated from infected people that are not recognized by the early immune response. Furthermore, genomic changes in the virus show host dependence and emerge to prominence with a temporal pattern that is consistent with selection for escape from an earlier immune response. Escape from immune recognition may therefore be a major factor in allowing persistent viral replication in HIV infection.     

Broadly Neutralizing Antibodies Developed by an HIV-Positive Elite Neutralizer Exact a Replication Fitness Cost on the Contemporaneous Virus

Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1⁺) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that in vivo escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.     

Evolution of human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitopes: fitness-balanced escape

CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.     

Selection for Neutralization Resistance of the Simian/Human Immunodeficiency Virus SHIVSF33A Variant In Vivo by Virtue of Sequence Changes in the Extracellular Envelope Glycoprotein That Modify N-Linked Glycosylation

We previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIVSF33, into a pathogenic biologic viral variant, designated SHIVSF33A. In the present study, we show that SHIVSF33A is resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout the env gene of SHIVSF33A; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIVSF33A, mutant viruses were generated on the backbone of parental SHIVSF33 and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIVSF33. The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.     

Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.     

Sequences in Glycoprotein gp41, the CD4 Binding Site,and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals.     

Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1.     

Length polymorphism within the second variable region of the human immunodeficiency virus type 1 envelope glycoprotein affects accessibility of the receptor binding site.

Sequential mutations were introduced into the V2 region of human immunodeficiency virus (HIV) type 1 HXB2, affecting the length, charge, and number of potential glycosylation sites. The insertions had no effect on cytopathicity or on the ability of virus to replicate in peripheral blood mononuclear cells and established T-cell lines. However, deletion of amino acids 186 to 188, encoding a conserved glycosylation site, resulted in a nonviable virus, suggesting a minimal length requirement of 40 amino acids for a functional V2 loop. However, all amino acid insertions affected the sensitivity of the variants to neutralization by soluble CD4 and monoclonal antibodies specific for epitopes in the V3 and CD4 binding site regions. Furthermore, these mutant viruses showed resistance to neutralization by HIV-positive human sera. Soluble gp120 mutant glycoproteins showed increased affinities for soluble CD4 and monoclonal antibodies specific for a number of epitopes overlapping the CD4 binding site, confirming that length increases in V2 affect exposure of the CD4 binding site. In summary, these data demonstrate that differences in V2 length modulate immunoreactivity of the envelope glycoprotein and support an association between the V2 and CD4 binding site regions.     

CD4-Induced Conformational Changes in the Human Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences for Virus Entry and Neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

Sequence variation of HIV and bioinformatics

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) interacts with receptors on the target cell and mediates virus entry by fusing the viral and cell membranes. To maintain the viral infectivity, amino acids that interact with receptors are expected to be more conserved than the other sites on the protein surface. In contrast to the functional constraint of amino acids for the receptor binding, some amino acid changes in this protein may produce antigenic variations that enable the virus to escape from recognition of the host immune system. Therefore, both positive selection (higher fitness) and negative selection (lower fitness) against amino acid changes are taking place during evolution of surface proteins of parasites To elucidate the evolutionary mechanisms of the whole HIV-1 gp120 envelope glycoprotein at the single site level, we collected and analyzed all available sequence data for the protein. By analyzing 186 sequences of the HIV-1 gp120 (subtype B), we reevaluated amino acid variability at the single site level, and estimated the numbers of synonymous and nonsynonymous substitutions at each codon position to detect positive and negative selection. We identified 33 amino acid positions which may be under positive selection. Some of these positions may form discontinuous epitopes. We also analyzed amino acid sequences to find amino acid positions responsible for usage of the second receptor. We found that, in addition to the V3 loop, amino acid variation at residue 440 in C4 region is clearly linked with the usage of CXCR 4.     

Reversal of human immunodeficiency virus type 1 IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker with a progressive clinical course

The role of humoral immunity in controlling human immunodeficiency virus type 1 (HIV-1) is still controversial. The resistance of primary HIV-1 variants to neutralization by antibodies, sera from HIV-1-infected patients, and soluble CD4 protein has been suggested to be a prerequisite for the virus to establish persistence in vivo. To further test this hypothesis, we studied the neutralization sensitivity of two IIIB/LAV variants that were isolated from a laboratory worker who accidentally was infected with the T-cell-line-adapted neutralization-sensitive IIIB isolate. Compared to the original virus in the inoculum, the reisolated viruses showed an increased resistance to neutralization over time. The ratio of nonsynonymous to synonymous nucleotide substitutions in the envelope gene pointed to strong positive selection. The emergence of neutralization-resistant HIV preceded disease development in this laboratory worker. Our results imply that the neutralization resistance of primary HIV may indeed be considered an escape mechanism from humoral immune control.     

Topographical rearrangements of visna virus envelope glycoprotein during antigenic drift.

Visna virus undergoes antigenic drift during persistent infection in sheep and thus eludes neutralizing antibodies directed against its major envelope glycoprotein, gp135. Antigenic variants contain point mutations in the 3' end of the genome, presumably within the envelope glycoprotein gene. To localize the changes in the viral proteins of antigenic mutants, we isolated 35 monoclonal antibodies (MAbs) against the envelope glycoprotein gp135 or the major core protein p27 of visna virus. The MAbs defined five partially overlapping epitopes on gp135. We used the MAbs and polyclonal immune sera directed against visna virus, gp135, or p27 in enzyme-linked immunosorbent assays to compare visna virus (strain 1514) with antigenic mutants (LV1-1 to LV1-6) previously isolated from a single sheep persistently infected with plaque-purified strain 1514. Polyclonal immune sera and anti-core p27 MAbs failed to distinguish antigenic differences among the viruses. By contrast, the anti-gp135 MAbs detected changes in all five epitopes of the envelope glycoprotein. Three gp135 epitopes, prominently exposed on strain 1514, were lost or obscured on the mutants; two covert gp135 epitopes, poorly exposed on strain 1514, were reciprocally revealed on the mutants. Even virus LV1-2, which is indistinguishable from parental strain 1514 by serum neutralization tests and which differs from it by only two unique oligonucleotides on RNase-T1 fingerprinting, displayed global changes in gp135. Our data suggest that visna virus variants may emerge more frequently during persistent infection than can be detected by serological tests involving the use of polyclonal immune sera, and the extent of phenotypic changes in their envelope glycoproteins may be greater than predicted by the small number of genetic changes previously observed. We suggest that topographical rearrangements in the three-dimensional structure of gp135 may magnify the primary amino acid sequence changes caused by point mutations in the env gene. This may complicate strategies to construct lentiviral vaccines by using the envelope glycoprotein.