Evolution of Envelope Sequences of Human Immunodeficiency Virus Type 1 in Cellular Reservoirs in the Setting of Potent Antiviral Therapy

In human immunodeficiency virus (HIV)-infected patients treated with potent antiretroviral therapy, the persistence of latently infected cells may reflect the long decay half-life of this cellular reservoir or ongoing viral replication at low levels with continuous replenishment of the population or both. To address these possibilities, sequences encompassing the C2 and V3 domains of HIV-1 env were analyzed from virus present in baseline plasma and from viral isolates obtained after 2 years of suppressive therapy in six patients. The presence of sequence changes consistent with evolution was demonstrated for three subjects and correlated with less complete suppression of viral replication, as indicated by the rapidity of the initial virus load decline or the intermittent reappearance of even low levels of detectable viremia. Together, these results provide evidence for ongoing replication. In the remaining three patients, virus recovered after 2 years of therapy was either genotypically contemporary with or ancestral to virus present in plasma 2 years before, indicating that virus recovery had indeed resulted from activation of latently infected cells.     

Dendritic Cell–T-Cell Interactions Support Coreceptor-Independent Human Immunodeficiency Virus Type 1 Transmission in the Human Genital Tract

Worldwide, human immunodeficiency virus (HIV) is transmitted predominantly by heterosexual contact. Here, we investigate for the first time, by examining mononuclear cells obtained from cervicovaginal tissue, the mechanisms whereby HIV type 1 (HIV-1) directly targets cells from the human genital tract. In contrast to earlier findings in mucosal models such as human skin, we demonstrate that the majority of T cells and macrophages but none or few dendritic cells (DC) express the HIV-1 coreceptor CCR5 in normal human cervicovaginal mucosa, whereas all three cell types express the coreceptor CXCR4. To understand the role of coreceptor expression on infectivity, mucosal mononuclear cells were infected with various HIV-1 isolates, using either CCR5 or CXCR4. Unstimulated T cells become rapidly, albeit nonproductively, infected with R5- and X4-tropic variants. However, DC and T cells form stable conjugates which permit productive infection by viruses of both coreceptor specificities. These results indicate that HIV-1 can exploit T-cell-DC synergism in the human genital tract to overcome potential coreceptor restrictions on DC and postentry blocks of viral replication in unactivated T cells. Thus, mononuclear cells infiltrating the genital mucosa are permissive for transmission of both R5- and X4-tropic HIV-1 variants, and selection of virus variants does not occur by differential expression of HIV-1 coreceptors on genital mononuclear cells.     

Clade-Specific Evolution Mediated by HLA-B*57/5801 in Human Immunodeficiency Virus Type 1 Clade A1 p24

HLA-B*57-mediated selection pressure leads to a typical escape pathway in human immunodeficiency virus type 1 (HIV-1) CD8 epitopes such as TW10. Whether this T242N pathway is shared by all clades remains unknown. We therefore assessed the nature of HLA-B*57 selection in a large, observational Kenyan cohort where clades A1 and D predominate. While T242N was ubiquitous in clade D HLA-B*57⁺ subjects, this mutation was rare (15%) in clade A1. Instead, P243T and I247L were selected by clade A1-infected HLA-B*57 subjects but not by HLA-B*5801⁺ subjects. Our data suggest that clade A1 consensus proline at Gag residue 243 might represent an inherent block to T242N escape in clade A1. We confirmed immunologically that P243T and I247L likely represent escape mutations. HLA-B*57 evolution also differed between clades in the KF11 and IW9 epitopes. A better understanding of clade-specific evolution is important for the development of HIV vaccines in regions with multiple clades.Human immunodeficiency virus type 1 (HIV-1) displays extreme genetic diversity, with nine clades (subtypes) described in group M, and frequent genomic recombination among and within the clades (7, 44). HIV is also capable of rapid evolution, which can lead to mutational escape from immune control (43). Escape from CD8⁺ T-cell responses occurs frequently in HIV-1 infection through mutations that affect epitope processing, HLA class I binding, and/or T-cell receptor recognition (23). In early HIV-1 infection, the majority of amino acid substitutions are associated HLA class I alleles (1). The timing and consequences of mutational escape from CD8⁺ T-cell responses vary considerably (8, 22).HLA-B*57, and to a lesser extent HLA-B*5801, has been associated with slower progression to AIDS in several studies (18, 27, 39), and HLA-B*5701 was associated with a lower viremia set point in a genome-wide association study (16). Several attributes of HLA-B*57-restricted CD8⁺ T-cell responses may contribute to their protectiveness, including dominant responses in acute infection (2), recognition of protective epitopes in HIV-1 p24 (33), better recognition of epitope variation (45), and retention of proliferative capability in chronic infection (24).HLA-B*57/5801 also exert powerful selection pressure on HIV to avoid CD8⁺ T-cell recognition. This was first demonstrated in the HLA-B*57-restricted TW10 epitope (TSTLQEQIGW [Gag_240-249]), which accounts for >30% of overall HIV-specific CD8⁺ T-cell responses in acutely infected HLA-B*57⁺ subjects (3). Escape in this epitope usually occurs early in infection, which coincidently is when HLA-B*57 is most protective (18). In clade B and C infections, >75 to 100% of HLA-B*57/5801⁺ subjects develop the T242N escape mutation, while HLA-B*57/5701-negative subjects rarely display polymorphism at this residue (5, 9, 10, 15, 32, 35, 38, 41). When T242N is transmitted to HLA-B*57/5801-negative subjects, it rapidly reverts to the consensus, suggesting that T242N is associated with a fitness defect (32, 35).While CD8⁺ T-cell cross-clade recognition has been tested extensively (6, 11, 19, 36, 48), few studies have addressed the possibility of clade-specific escape from CD8⁺ T-cell responses. This may be especially relevant where clade consensus sequences differ in immunologically relevant epitopes. Here we demonstrate in a large Kenyan cohort substantial differences in HLA-B*57/B*5801-mediated selection among HIV clades.Participants were enrolled from a Nairobi, Kenya-based cohort, and the relevant ethical review boards approved the study. HLA typing was performed as described previously (34). CD4 counts were measured longitudinally at biannual visits. Multiple and other clade infections were excluded. The HIV-1 p24 gene was amplified from proviral HIV DNA or RNA using a nested PCR approach and sequenced, and viral subtyping was carried out as described previously (42). Previously described HLA-B*57 epitopes IW9 (ISPRTLNAW), KF11 (KAFSPEVIPMF), and TW10 (TSTLQEQIGW) and selected variants were tested in immunological assays and described where relevant. Gamma interferon enzyme-linked immunospot (ELISPOT) assays were performed as described previously (37) using blood samples from HLA-B*57⁺ and -B*5801⁺ subjects. All peptides were tested at concentrations of 10 μg, 1 μg, 0.1 μg, and 0.01 μg/ml. Responses were considered positive if they were more than two times higher than that of the negative control and were measured at ≥100 spot-forming units ml⁻¹. Fisher''s exact test and chi-square analyses were used to determine differences among groups in categorical analyses. Mann-Whitney U tests were used to compare response magnitudes and disease progression between groups.We confirmed the protective effects of HLA-B*57 in clade A1 infection (mean of 9.9 years versus 7.8 years until CD4 counts were <200, P = 0.041) (Fig. ​(Fig.1).1). Slow progressors were overrepresented in HLA-B*57⁺ clade A1⁺ subjects (52.2%) compared to both HLA-B*5801⁺ clade A1⁺ (13.3%, P = 0.02) and HLA-B*57/5801-negative clade A1⁺ (27.8%, P = 0.028) subjects (Fig. ​(Fig.1b).1b). In contrast to what has been shown for other clades (2, 27), protection was not observed for clade A1-infected HLA-B*5801⁺ subjects (mean of 6.5 years versus 7.8 years until CD4 counts were <200, P > 0.3) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.HLA-B*57, but not HLA-B*5801, is associated with a lower rate of disease progression in clade A1-infected subjects than that of the overall cohort. (a) Number of years from cohort entry until sequential CD4 counts fell below 200/μl. (b) Slow progressors (>10 years with CD4 counts of >200) were also more common in HLA-B*57⁺ clade A1-infected subjects than in those expressing HLA-B*5801 or neither.Stratification of TW10 (Gag_240-249) proviral sequences on the basis of HLA allele and clade revealed several differences in selection between clades A1 and D (Fig. ​(Fig.2a).2a). We observed the expected T242N substitution in 100% of HLA-B*57⁺ clade D-infected subjects (7/7), compared to only 14.7% variability at Gag residue 242 in HLA-B*57/5801-negative subjects (13/88, P = 3.26 × 10⁻⁹) (Fig. ​(Fig.2b).2b). In contrast, T242N was found infrequently in clade A1-infected HLA-B*57⁺ subjects (15%, 5/33, P = 0.0004). Instead, variants containing the mutations P243T and I247L were more frequently observed (both observed in 11/33 subjects). Overall, variation at residues 243 and 247 was more common in HLA-B*57⁺ subjects (51% and 15%, respectively; P = 2.92 × 10⁻⁶) than in HLA-B*57/5801-negative clade A1⁺ subjects (33% and 9%, respectively; P = 0.0008). Selection at both residues 243 and 247 was observed only in 2/33 HLA-B*57⁺ subjects, suggesting that these mutations are independent. Selection at residue 248, observed in clade B infection (32), was not evident in either clade A1 or D. While I247X selection has been described in other clades at low frequencies and in elite controllers (21, 40), HLA-B*57-mediated selection at Gag residue 243 has not yet been described. In summary, the T242N mutation, which is typical of other clades, does not appear to be the primary escape mutant in clade A1.Open in a separate windowFIG. 2.HLA-B*57-mediated selection in TW10 differs between clade A1 and clade D. (a) TW10 sequences were stratified by HLA-B*57, HLA-B*5801, or other alleles (HLA-B*57/5801⁻) and compared between clades A1 and D, based on the clade B consensus TW10 sequence. Each subject is represented by one sequence, and the numbers of subjects with a given sequence are shown in parentheses. A summary of variation from the TW10 consensus at Gag residues 242, 243, 247, and 248 is shown for HLA-B*57⁺ (b) and -B*5801⁺ (c) subjects. (b and c) Clade D is shown at the top, and clade A1 is shown at the bottom. Variation is shown in dark gray, and consensus is shown in light gray. (d) The proportions of clade A1-infected subjects with selection at Gag residue 242 only, and those with selection at residues 242 and 243 in combination, are shown. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.Previous studies have suggested that HLA-B*5801 places selection pressure on TW10, similar to that of HLA-B*57 (35). Similar to clades B and C, selection of T242N was evident in HLA-B*5801⁺ clade D-infected subjects (TW10 variation in 8/11 HLA-B*5801⁺ subjects versus 13/88 HLA-B*57/5801-negative subjects; P = 0.0069) (Fig. ​(Fig.2c).2c). Limited T242N selection was observed in clade A1-infected HLA-B*5801⁺ subjects, and in contrast to HLA-B*57, there were no HLA-B*5801-associated substitutions at residues 243 and 247 in clade A1 (P values of 0.75 and 0.29, respectively) (Fig. ​(Fig.2c).2c). In summary, these data suggest that in addition to HLA-B*5801 not being associated with protection in clade A1 (Fig. ​(Fig.1),1), HLA-B*5801 does not select the HLA-B*57-associated clade A1 TW10 escape mutations.Inclusion of all clade A1 sequences with T242X substitutions (regardless of the HLA allele) reveals that in every case (10/10), there is an accompanying residue 243 mutation. Polymorphisms at these sites correlate very strongly (P = 3.71 × 10⁻⁸) (Fig. ​(Fig.2d).2d). Together, these data suggest that residue 242 polymorphism in clade A1 is incompatible with proline at residue 243, which is the clade A1 consensus.We next assessed the immunological implications of novel clade A1 variants in HLA-B*57⁺ (n = 12) and -B*5801⁺ (n = 6) subjects infected primarily by clade A1. Clade A1-infected subjects commonly made anamnestic, low-avidity responses to TW10. The majority of HLA-B*57⁺ subjects who recognized clade A1 TW10 did not respond to P243T or I247L in ELISPOT assays (Fig. ​(Fig.3),3), supporting the hypothesis that these represent escape mutations. Those who did recognize P243T and I247L had lower magnitude responses than those who recognized clade A1 TW10 at the 10-μg/ml peptide concentration (P of 0.0005 for both) (Fig. ​(Fig.3a).3a). Similarly, these variants were not well recognized by CD8⁺ T cells from HLA-B*5801⁺ subjects (Fig. ​(Fig.3b).3b). For two HLA-B*57⁺ subjects, P243T and I247L responses had lower avidity than clade A1 TW10 responses (Fig. ​(Fig.3c).3c). These data support the hypothesis that P243T and I247L likely represent escape mutations.Open in a separate windowFIG. 3.Peptides with novel TW10 clade A1-selected mutations are poorly recognized in ex vivo gamma interferon ELISPOT avidity assays, suggestive of escape mutations. ELISPOT responses to TW10 and variants at 10 μg/ml peptide by HLA-B*57⁺ (a) and HLA-B*5801⁺ (b) subjects are shown. (c) The functional avidity of TW10 and variants for two HLA-B*57⁺ subjects is shown, suggesting that P243T and I247L are less recognized than TW10, particularly at lower peptide concentrations. Sequence names are described in the text. (d) Elispot responses to A1 TW10 and T242N correlated at 10 μg/ml. SFU/m, spot-forming units/million PBMCs.Recognition of the clade B/D consensus (TSTLQEQIGW) was diminished compared to that of clade A1 TW10. However, despite the presumed absence of this variant in these subjects'' autologous sequences, the clade B/D escape variant (TSNLQEQIGW [T242N]) was recognized at a magnitude similar to that of the consensus clade A1 TW10 (r = 0.71, P = 0.0099) (Fig. ​(Fig.3d).3d). No responses to T242N/G248A were observed (not shown), as described previously (32). These data suggest that clade A1 and B TW10, and their escape variants, are immunologically distinct from one another.We next assessed whether clade-specific selection was evident in other immunodominant HLA-B*57 p24 epitopes that are commonly targeted in chronic clade B infection (2). In clade D IW9 (ISPRTLNAW [Gag_147-155]), variants containing the escape variant I147L (14) were more common in HLA-B*57⁺ subjects than in HLA-B*57/5801-negative subjects (86% and 30%, respectively; P = 0.0055) (Table ​(Table1).1). However, this variant was not selected in clade A1 (variation in 30% versus 21% subjects; P value was not significant), where leucine is the consensus. Interestingly, ELISPOT data indicated substantial cross-reactivity between 147L and 147I in clade A1-infected subjects (10 μg/ml, r = 0.987, P < 0.0001) (data not shown), suggesting that infection with an escape variant from one clade (clade D) does not necessarily preclude recognition of this epitope in another one (clade A1). Other amino acids (F, M, and P) were common in HLA-B*57⁺ subjects at residue 147 (>30% versus 3% in HLA-B*57/5801-negative subjects, P = 3.85 × 10⁻⁶). Although the immunological consequences are unknown, these HLA-associated substitutions could represent novel escape variants.

TABLE 1.

p24 sequences in HLA-B*57⁺ subjects infected by clades A1 and D

A Conformation-Specific Monoclonal Antibody Reacting with Fusion-Active gp41 from the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays a major role in the membrane fusion step of viral infection. The ectodomain of gp41 contains a six-helix structural domain that likely represents the core of the fusion-active conformation of the molecule. A monoclonal antibody (MAb), designated NC-1, was generated and cloned from a mouse immunized with the model polypeptide N36(L6)C34, which folds into a stable six-helix bundle. NC-1 binds specifically to both the α-helical core domain and the oligomeric forms of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the gp41 core. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4. These results indicate that NC-1 is capable of reacting with fusion-active gp41 in a conformation-specific manner and can be used as a valuable biological reagent for studying the receptor-induced conformational changes in gp41 required for membrane fusion and HIV-1 infection.     

Human Immunodeficiency Virus Type 1 Populations in Blood and Semen

Transmission of human immunodeficiency virus type 1 (HIV-1) usually results in outgrowth of viruses with macrophage-tropic phenotype and consensus non-syncytium-inducing (NSI) V3 loop sequences, despite the presence of virus with broader host range and the syncytium-inducing (SI) phenotype in the blood of many donors. We examined proviruses in contemporaneous peripheral blood mononuclear cells (PBMC) and nonspermatozoal semen mononuclear cells (NSMC) of five HIV-1-infected individuals to determine if this preferential outgrowth could be due to compartmentalization and thus preferential transmission of viruses of the NSI phenotype from the male genital tract. Phylogenetic reconstructions of ~700-bp sequences covering the second constant region through the fifth variable region (C2 to V5) of the viral envelope gene revealed distinct variant populations in the blood versus the semen in two patients with AIDS and in one asymptomatic individual (patient 613), whereas similar variant populations were found in both compartments in two other asymptomatic individuals. Variants with amino acids in the V3 loop that predict the SI phenotype were found in both AIDS patients and in patient 613; however, the distribution of these variants between the two compartments was not consistent. SI variants were found only in the PBMC of one AIDS patient but only in the NSMC of the other, while they were found in both compartments in patient 613. It is therefore unlikely that restriction of SI variants from the male genital tract accounts for the observed NSI transmission bias. Furthermore, no evidence for a semen-specific signature amino acid sequence was detected.     

Soluble CD23 in Human Immunodeficiency Virus Type 1 Infected Patients

The level of sCD23 produced in the course of human immunodeficiency virus (HIV) infection was measured in patients grouped according to the Centers for Disease Control by using an immunoradiometric assay. Soluble CD23 was evaluated in supernatants of peripheral blood mononuclear cell (PBMC) (10⁶ cells/ml) stimulated by phytohemagglutinin (PHA). Compared with healthy controls (m±S.D. = 1.0 ±0.34 U/ml, n = 7), higher values were observed in some of the patients of group II (asymptomatic) (m±S.D. = 2±1.33, n = 9) and some of the patients of group IV (AIDS) (m±S.D. = 1.3 ±1.40, n = 8). Those results prompted us to compare the plasma levels of sCD23 in group II and group IV HIV-infected patients and in healthy individuals. Soluble CD23 plasma levels in healthy patients (n = 42) ranged from 0 to 1.5 U/ml (m±S.D. = 0.9±0.33), in group II patients (n = 17) from 0 to 3 U/ml (m±S.D. = 0.92±0.83) and in group IV patients (n =73) from 0 to 2.9 U/ml (m±S.D. = 1.15±0.71). The differences between the patients and the healthy individuals were not statistically significant but individual sCD23 values higher than 2 U/ml were obtained in 6% of the group II patients and 16.7% of the group IV patients. Increased values of sCD23 were obtained in plasma from patients with secondary infectious diseases (groups IV-C1 and IV-C2) and from patients without secondary infectious diseases (group II, group IV-A and group IV-B). Elevated values of sCD23 were detected even in patients with low counts of CD4⁺ T cells and CD8⁺ T cells in their peripheral blood. sCD23 has numerous activities including control of IgE synthesis and cytokine-like properties. Our results show a disarray of sCD23 in HIV-infected patients which could be involved in drug reactions, allergic manifestations and the IgE-level increase. Further investigations should attempt to define the role of sCD23 in clinical manifestations of HIV infection.     

Neutralizing Antibodies in Sera from Macaques Infected with Chimeric Simian-Human Immunodeficiency Virus Containing the Envelope Glycoproteins of either a Laboratory-Adapted Variant or a Primary Isolate of Human Immunodeficiency Virus Type 1

The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1.     

Dynamics of Human Immunodeficiency Virus Type 1 Replication in Vertically Infected Infants

Plasma human immunodeficiency virus type 1 (HIV-1) turnover and kinetics were studied in children aged 15 days to 2 years following the initiation of a triple antiretroviral drug regimen consisting of zidovudine, lamivudine, and nevirapine. HIV-1 turnover was at least as rapid as that previously described in adults; turnover rates were more rapid in infants and children aged 3 months to 2 years than in infants less than 3 months of age. These data confirm the central role of HIV-1 replication in the pathogenesis of vertical HIV-1 infection and reinforce the importance of early, potent combination therapies for the long-term control of HIV-1 replication.     

Widespread Adaptive Evolution in the Human Immunodeficiency Virus Type 1 Genome

We investigated variable selective pressures among amino acid sites in HIV-1 genes. Selective pressure at the amino acid level was measured by using the nonsynonymous/synonymous substitution rate ratio ( = d_N/d_S). To identify amino acid sites under positive selection with > 1, we applied maximum likelihood models that allow variable ratios among sites to analyze genomic sequences of 26 HIV-1 lineages including subtypes A, B, and C. Likelihood ratio tests detected sites under positive selection in each of the major genes in the genome: env, gag, pol, vif, and vpr. Positive selection was also detected in nef, tat, and vpu, although those genes are very small. The majority of positive selection sites is located in gp160. Positive selection was not detected if was estimated as an average across all sites, indicating the lack of power of the averaging approach. Candidate positive selection sites were mapped onto the available protein tertiary structures and immunogenic epitopes. We measured the physiochemical properties of amino acids and found that those at positive selection sites were more diverse than those at variable sites. Furthermore, amino acid residues at exposed positive selection sites were more physiochemically diverse than at buried positive selection sites. Our results demonstrate genomewide diversifying selection acting on the HIV-1.     

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.     

Variants from the Diverse Virus Population Identified at Seroconversion of a Clade A Human Immunodeficiency Virus Type 1-Infected Woman Have Distinct Biological Properties

Development of effective therapeutics to prevent new infections with human immunodeficiency type 1 (HIV-1) is predicated on an understanding of the properties that provide a selective advantage to a transmitted viral population. In contrast to the homogeneous virus population that typifies early HIV-1 infection of men, the viral population in women recently infected with clade A HIV-1 is genetically diverse, based on evaluation of the envelope gene. A longitudinal study of viral envelope evolution in several women suggested that representative envelope variants detected at seroconversion had distinct biological properties that affected viral fitness. To test this hypothesis, a full-length, infectious molecular clone, Q23-17, was obtained from an infected woman 1 year following seroconversion, and chimeric viruses containing envelope genes representative of seroconversion and 27-month-postseroconversion populations were constructed. Dendritic cells (DC) could transfer infection of seroconversion variant Q23ScA, which dominated the viral population in the year following seroconversion, and the closely related 1-year isolate Q23-17 to resting peripheral blood mononuclear cells (PBMC). In contrast, resting PBMC exposed to DC pulsed with Q23ScB, which was detected infrequently in samples after seroconversion, or the 27-month chimeras were inconsistently infected. Additionally, quiescent PBMC infected with Q23ScA or Q23-17 proliferated more robustly than uninfected cells or cells infected with the other envelope chimeras in response to immobilized anti-CD3. Stimulation with tetanus toxoid led to an increased proportion of CD45RA+ cells and a decreased expression of CD28 on CD45RO+ cells in cultures of Q23-17-infected PBMC. These data demonstrate that variants from the heterogeneous seroconversion clade A HIV-1 population in a Kenyan woman have distinct biological features that may influence viral pathogenesis.     

Cytotoxic T Cells from Human Immunodeficiency Virus Type 2-Infected Patients Frequently Cross-React with Different Human Immunodeficiency Virus Type 1 Clades

Knowledge of immune mechanisms responsible for the cross-protection between highly divergent viruses such as human immunodeficiency virus type 1 (HIV-1) and HIV-2 may contribute to an understanding of whether virus variability may be overcome in the design of vaccine candidates which are broadly protective across the HIV subtypes. We demonstrate that despite the significant difference in virus amino acid sequence, the majority of HIV-2-infected individuals with different HLA molecules possess a dominant cytotoxic T-cell response which is able to recognize HIV-1 Gag protein. Furthermore, HLA-B5801-positive subjects show broad cross-recognition of HIV-1 subtypes since they mounted a T-cell response that tolerated extensive amino acid substitutions within HLA-B5801-restricted HIV-1 and HIV-2 epitopes. These results suggests that HLA-B5801-positive HIV-2-infected individuals have an enhanced ability to react with HIV-1 that could play a role in cross-protection.Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are related human retroviruses that show various biological and structural differences. HIV-2 is found mainly in West Africa, whereas HIV-1 is spreading throughout the world. HIV-2 is less transmissible, and HIV-2-positive patients exhibit longer clinical latency periods than individuals infected with HIV-1 (23). A recent report has also shown that the mortality in HIV-2-infected individuals is only twice as high as in the uninfected population and, in the majority of adults, survival is not affected by HIV-2 status (31).Although the two viruses are similar in genomic organization, various genetic and enzymatic differences have been found at many stages of the retroviral life cycle. They differ significantly in terms of amino acid sequence, the more conserved being the Pol and Gag sequences, which exhibit less than 60% homology (17).Despite these differences, epidemiological data and animal studies have shown some evidence of cross-protection between the two viral infections. Travers et al. reported that HIV-2-infected women had a lower incidence of HIV-1 infection than did HIV-seronegative women in a cohort of commercial sexual workers in Dakar (37), and rhesus macaques immunized with a recombinant HIV-1 poxvirus vaccine are protected against HIV-2 challenge (2). These studies, though not conclusive (1, 6), suggest that differences in the virus may not necessarily preclude the development of defensive immunity to a subsequent pathogenic infection, an old-fashioned concept pioneered by Jenner, who used cowpox to vaccinate against human smallpox.The immunological basis of cross-protection is largely unknown, and a clear understanding of the role played by the humoral or cell-mediated immune response in HIV protection is still lacking. However, mounting evidence suggests that cytotoxic T-lymphocyte (CTL) response could be the key element. Indeed, the protection afforded in animal models against simian (13) and feline (12) immunodeficiency virus infections is closely correlated with the induction of specific CTL response, and HIV-1 and HIV-2 HLA-B35-restricted cross-reactive CTLs have been postulated to confer protection against repeated HIV exposure (33).CTLs recognize short viral peptides, 8 to 11 amino acids long, that are generated by the intracellular processing of endogenously synthesized viral antigens within the infected cells, which are expressed at the cell surface in the binding groove of HLA class I molecules. The specificity of the T-cell response is determined by the interaction of the antigen-specific T-cell receptor (TCR) with the peptide-HLA complex, and this interaction, together with non-antigen-specific signals, activates the CTLs (15).The presence of cross-reactive CTLs able to lyse HIV-1- or HIV-2-infected cells should be dependent on the extent of conservation between the two viruses within the epitopes selected by particular HLA class I molecules. It is well known that amino acid substitutions within the epitopes can abrogate the CTL response by inhibiting either HLA binding or TCR recognition (32). However, a number of recent studies have shown that T cells can recognize apparently unrelated peptides (10, 41), and crystallographic data have shown physical limits to the TCR epitope specificity due to the limited size of contact between the TCR and the peptide (14), suggesting a flexibility in T-cell recognition of antigen (19).Some individuals with a particular HLA profile which is responsible for presentation of the viral antigen and for selection of the T-cell repertoire may possess a CTL response not affected by mutations within the epitope, as has been demonstrated in subjects with HLA alleles B27 (28) and B35 (33). In these cases, amino acid substitutions within the HIV-1 and -2 epitopes were tolerated by the CTLs.In this study, we have investigated the extent of cross-reacting CTLs between HIV-2 and HIV-1 in a group of HIV-2-infected subjects with different HLA class I types. We have shown that despite differences in amino acid sequence between the two viruses, the majority of HIV-2-positive subjects possess CTLs which are able to recognize HIV-1 Gag protein.Furthermore, analysis of HLA profiles and the fine specificity of the cytotoxic response demonstrated that HLA-B5801-positive subjects show broad cross-recognition of HIV-1 isolates. These subjects mounted a CTL response that tolerated extensive amino acid substitutions within an HLA-B5801-restricted HIV-1 epitope.     

An Envelope Modification That Renders a Primary, Neutralization-Resistant Clade B Human Immunodeficiency Virus Type 1 Isolate Highly Susceptible to Neutralization by Sera from Other Clades

SF162 is a primary (PR), non-syncytium-inducing, macrophagetropic human immunodeficiency virus type 1 (HIV-1) clade B isolate which is resistant to antibody-mediated neutralization. Deletion of the first or second hypervariable envelope gp120 region (V1 or V2 loop, respectively) of this virus does not abrogate its ability to replicate in peripheral blood mononuclear cells and primary macrophages, nor does it alter its coreceptor usage profile. The mutant virus with the V1 loop deletion, SF162ΔV1, remains as resistant to antibody-mediated neutralization as the wild-type virus SF162. In contrast, the mutant virus with the V2 loop deletion, SF162ΔV2, exhibits enhanced susceptibility to neutralization by certain monoclonal antibodies whose epitopes are located within the CD4-binding site and conserved regions of gp120. More importantly, SF162ΔV2 is now up to 170-fold more susceptible to neutralization than SF162 by sera collected from patients infected with clade B HIV-1 isolates. In addition, it becomes susceptible to neutralization by sera collected from patients infected with clade A, C, D, E, and F HIV-1 isolates. These findings suggest that the V2, but not the V1, loop of SF162 shields an as yet unidentified region of the HIV envelope rich in neutralization epitopes and that the overall structure of this region appears to be conserved among clade B, C, D, E, and F HIV-1 PR isolates.     

Differences in Variation of Human Immunodeficiency Virus Type 1 Sequences from Henan and Shanghai Regions of China

Illegally paid blood donation was a risk factor for HIV acquisition exclusively in Henan and Hubei Provinces of China,and not in Shanghai.Nucleotide sequences in the gag and env genes of HIV-1 were compared between isolates from Henan and Shanghai regions of China to test whether an expected higher degree of a common source of infections from this unique blood donation transmission risk would be evident as decreased variation among Henan isolates in an exploratory cross-sectional analysis.Among 38 isolates studied,23 of 23(100%)from Henan and 8 of 15(54%)from Shanghai were subtype B.In addition,fewer sequence differences were found in gp41 of subtype B isolates from Henan than from Shanghai isolates.Further studies with additional controls are therefore warranted to confirm the role of the degree of a common source of infections in differences in HIV variation across populations.     

Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.     

Expanded Breadth of the T-Cell Response to Mosaic Human Immunodeficiency Virus Type 1 Envelope DNA Vaccination

An effective AIDS vaccine must control highly diverse circulating strains of human immunodeficiency virus type 1 (HIV-1). Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. One-, two-, and three-mosaic sets that increased theoretical epitope coverage were developed. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to those for natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.The development of AIDS vaccines has been advanced recently by demonstrations of increased survival and decreased viral load following vaccination with T-cell vaccines in nonhuman primate models (12, 19, 23, 26, 31, 37). Although such vaccine studies have implied that T cells may contribute to the control of viremia in the highly lethal simian immunodeficiency virus SIVmac251 challenge model, the applicability of these results in human studies remains uncertain. The major concern regarding the efficacy of human immunodeficiency virus (HIV) vaccines in humans is the extraordinary genetic diversity of the virus. The sequence similarity of HIV type 1 (HIV-1) envelope from diverse isolates within a clade can diverge by as much as 15%, and divergence between alternative clades may approach 30% (10). In addition, the diversity of the viral Gag gene product can approach similar levels, particularly in p17 and p15, which are much more diverse than p24 (6), although Gag does not have the extreme localized diversity seen in the highly variable regions of Env (6, 10). While the approach to viral diversity has been addressed in existing vaccines through the use of envelopes derived from representative viruses in the major clades, increasing knowledge about the genetic diversity of naturally occurring isolates has enabled alternative approaches that enhance population coverage of vaccine-elicited T-cell responses.Approaches under consideration include the use of central gene sequences based on ancestral, consensus, or center-of-the-tree genetic analyses (5, 10, 18, 31, 36). Such prototypes are derived by selection of the most common amino acids at each residue (10, 16, 17, 21, 25, 36), identifying the most recent common ancestor of diverging viruses in a vaccine target population (5, 10, 18, 36), or modeling the sequence at the center of the phylogenetic tree (29), respectively. Peptides based on any of these three centralized protein strategies enhanced the detection of T-cell responses in natural infection relative to the use of peptides based on natural strains; however, all three strategies behaved equivalently (7).The use of a single M group consensus/ancestral Env sequence has been shown to elicit T-cell responses with greater breadth of cross-reactivity than single natural strains in animal models (31, 36). Such central sequences do not exist in nature, and even phylogenetic ancestral reconstructions are just an approximate model of an ancestral state of the virus (8). Thus, central sequence strategies have provided evidence that various informatically derived gene products can elicit immune responses to T-cell epitopes found in diverse circulating strains, leading to the possibility of using computational strategies to design polyvalent vaccines which optimize T-cell coverage (6, 24). In this study, we have evaluated for the first time the ability of nonnatural mosaic Env immunogens (6) to elicit T-cell responses of increased cross-reactivity against epitopes represented in naturally circulating viruses in animals.Mosaic HIV-1 envelope genes were derived using an informatic approach, whereby in silico-generated recombinants of natural variants from the Los Alamos database M group Env alignment were created, scored, and selected in combination to optimize the coverage of 9-mers in the global database for a given vaccine cocktail size. While mosaic proteins are artificial constructs that do not occur in nature, they align well to natural proteins, and any short span found in mosaics will tend to be found repeatedly among natural strains (although some of the hypervariable loop regions of Env are so extremely variable that they are not repeated among circulating strains, and this necessitates bridging these regions with segments found in a single strain). In silico recombination breakpoints are constrained to create fusion points found in natural sequences. It is possible to provide increased breadth of coverage with a single mosaic, providing the maximum possible single-antigen diversity coverage for stretches of nine amino acids. Alternatively, multiple mosaics can increase the breadth of representation but have the drawback of requiring the synthesis of additional vectors for clinical use. Mosaics also preserve a natural Env-like sequence to retain normal antigen processing. Here, we have compared single-, double-, or triple-mosaic envelope antigen sets to naturally circulating strains or other derivatives for their ability to elicit immune responses of increased breadth. The data suggest that mosaic HIV-1 envelope sequences provide an approach that may be useful in the development of HIV vaccines that respond to T-cell epitopes represented in naturally circulating strains.