Lack of Viral Escape and Defective In Vivo Activation of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes in Rapidly Progressive Infection

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses over the course of rapidly progressive infection are not well defined. Detailed longitudinal analyses of neutralizing antibodies, lymphocyte proliferation, in vivo-activated and memory cytotoxic T-lymphocyte (CTL) responses, and viral sequence variation were performed on a patient who presented with acute HIV-1 infection, developed an AIDS-defining illness 13 months later, and died 45 months after presentation. Neutralizing-antibody responses remained weak throughout, and no HIV-1-specific lymphocyte proliferative responses were seen even early in the disease course. Strong in vivo-activated CTL directed against Env and Pol epitopes were present at the time of the initial drop in viremia but were quickly lost. Memory CTL against Env and Pol epitopes were detected throughout the course of infection; however, these CTL were not activated in vivo. Despite an initially narrow CTL response, new epitopes were not targeted as the disease progressed. Viral sequencing showed the emergence of variants within the two targeted CTL epitopes; however, viral variants within the immunodominant Env epitope were well recognized by CTL, and there was no evidence of viral escape from immune system detection within this epitope. These data demonstrate a narrowly directed, static CTL response in a patient with rapidly progressive disease. We also show that disease progression can occur in the presence of persistent memory CTL recognition of autologous epitopes and in the absence of detectable escape from CTL responses, consistent with an in vivo defect in activation of CTL.     

Longitudinal Phenotypic Analysis of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes: Correlation with Disease Progression

Few studies have examined longitudinal changes in human immunodeficiency virus type 1 (HIV)-specific cytotoxic T lymphocytes (CTL). To more closely define the natural history of HIV-specific CTL, we used HLA-peptide tetrameric complexes to study the longitudinal CD8(+) T-cell response evolution in 16 A*0201-positive untreated individuals followed clinically for up to 14 years. As early as 1 to 2 years after seroconversion, we found a significant association between high frequencies of A*0201-restricted p17(Gag/Pol) tetramer-binding cells and slower disease progression (P < 0.01). We observed that responses could remain stable over many months, but any longitudinal changes that occurred were typically accompanied by reciprocal changes in RNA viral load. Phenotypic analysis with markers CD45RO, CD45RA, and CD27 identified distinct subsets of antigen-specific cells and the preferential loss of CD27(+) CD45RO(+) cells during periods of rapid decline in the frequency of tetramer-binding cells. In addition we were unable to confirm previous studies showing a consistent selective loss of HIV-specific cells in the context of sustained Epstein-Barr virus-specific cell frequencies. Overall, these data support a role of HIV-specific CTL in the control of disease progression and suggest that the ultimate loss of such CTL may be preferentially from the CD27(+) CD45RO(+) subset.     

HIV-1附属蛋白特异性细胞毒性T细胞应答研究

人类免疫缺陷病毒(Humanimmunodeficiencyvirus,HIV)附属蛋白Nef、Vpu、Vpr和Vif在病毒复制中起着关键作用,并能被细胞毒性T细胞(CytotoxicTLymphocyte,CTL)识别。然而,对我国HIV感染者体内附属蛋白特异性的CTL应答研究比较少。本研究应用覆盖HIV-1B、C亚型附属蛋白(Nef、Vpu、Vpr和Vif)的142个肽段作为抗原,通过酶联免疫斑点实验(Enzyme-LinkedImmunospot,ELISPOT)检测61例中国HIV/AIDS患者和10例HIV-1血清阴性对照的HIV-1附属蛋白特异性CTL应答。无论对HIV-1B亚型还是HIV-1C亚型附属蛋白都能产生特异性CTL应答,特别是Nef区蛋白的反应频率和累积应答强度都较高(P<0.001),B、C亚型间的应答频率和累积应答强度都无显著差别(P>0.05),其免疫优势区也大致相同。附属蛋白特异性的累积CTL应答强度将近达到总应答的21%。这些结果表明尽管HIV-1附属蛋白的体积小,但它们在诱导特异性的CTL应答中发挥了重要作用,对评价HIV-1免疫应答的幅度和特异性以及研发针对中国人群的HIV疫苗有重要的意义。     

HIV-1附属蛋白特异性细胞毒性T细胞应答研究

人类免疫缺陷病毒(Human immunodeficiency virus, HIV)附属蛋白Nef、Vpu、Vpr和Vif 在病毒复制中起着关键作用,并能被细胞毒性T细胞(Cytotoxic T Lymphocyte, CTL)识别.然而,对我国HIV感染者体内附属蛋白特异性的CTL应答研究比较少.本研究应用覆盖HIV-1B、C亚型附属蛋白(Nef、Vpu、Vpr和Vif)的142个肽段作为抗原,通过酶联免疫斑点实验(Enzyme-Linked Immunospot,ELISPOT)检测61例中国HIV/AIDS患者和10例HIV-1血清阴性对照的HIV-1附属蛋白特异性CTL应答.无论对HIV-1B 亚型还是HIV-1C亚型附属蛋白都能产生特异性CTL 应答,特别是Nef区蛋白的反应频率和累积应答强度都较高(P＜0.001),B、C亚型间的应答频率和累积应答强度都无显著差别(P＞0.05),其免疫优势区也大致相同.附属蛋白特异性的累积CTL应答强度将近达到总应答的21%.这些结果表明尽管HIV-1附属蛋白的体积小,但它们在诱导特异性的CTL应答中发挥了重要作用,对评价HIV-1免疫应答的幅度和特异性以及研发针对中国人群的HIV疫苗有重要的意义.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Strong Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T-Lymphocyte Activity in Sydney Blood Bank Cohort Patients Infected with nef-Defective HIV Type 1

Proposals for the use of live attenuated human immunodeficiency virus (HIV) type 1 (HIV-1) as a vaccine candidate in humans have been based on the protection afforded by attenuated simian immunodeficiency virus in the macaque model. Although it is not yet known if this strategy could succeed in humans, a study of the Sydney Blood Bank Cohort (SBBC), infected with an attenuated HIV-1 quasispecies with natural nef and nef/long terminal repeat deletions for up to 17 years, could provide insights into the long-term immunological consequences of living with an attenuated HIV-1 infection. In this study, HIV-specific cytoxic T-lymphocyte (CTL) responses in an SBBC donor and six recipients were examined over a 3-year period with enzyme-linked immunospot, tetrameric complex binding, direct CTL lysis, and CTL precursor level techniques. Strong HIV-specific CTL responses were detected in four of seven patients, including one patient with an undetectable viral load. Two of seven patients had weak CTL responses, and in one recipient, no HIV-specific CTLs were detected. High levels of circulating effector and memory HIV-specific CTLs can be maintained for prolonged periods in these patients despite very low viral loads.     

Antiviral Activity of the Proteasome on Incoming Human Immunodeficiency Virus Type 1

Following cell surface receptor binding and membrane fusion, human immunodeficiency virus (HIV) virion cores are released in the cytoplasm. Incoming viral proteins represent potential targets for cytosolic proteases. We show that treatment of target cells with the proteasome inhibitors MG132 and lactacystin increased the efficiency of HIV infection. Proteasome inhibitors were active at the early steps of the viral cycle. Incoming p24^Gag proteins accumulated in the cytosol, and larger amounts of proviral DNA were synthesized. In vitro, purified 20S proteasome degraded HIV virion components. Thus, degradation of incoming viral proteins by the proteasome represents an early intracellular defense against infection.     

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.     

Distinct Recognition of Non-Clade B Human Immunodeficiency Virus Type 1 Epitopes by Cytotoxic T Lymphocytes Generated from Donors Infected in Africa

We present detailed studies of human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) responses to clade A or C HIV type 1 in three donors infected in East Africa. We define several novel non-clade B CTL epitopes, including some restricted by HLA alleles common in Africans. Although cross-clade CTL recognition of these epitopes does occur, recognition can also be highly clade specific.     

In Vitro Modification of Human Immunodeficiency Virus Type 1 (HIV-1) Infectivity by the U937 Cells

The effect of host cell factors on infectivity of human immunodeficiency virus type 1 (HIV-1) was studied by infecting a monoblastoid cell line (U937) or a T-cell line (MOLT-4) with a highly infective single clone of HIV-1 and comparing the infectivity of the produced viruses to different cell lines. Chronically infected U937 cells consistently produced viruses with minimal infectivity. This phenotypic change was host-dependent as the back-passage of the U937-produced low infective viruses into MOLT-4 cells resulted in regaining their original high infectivity. Southern and Northern blot analyses of the HIV-1 grown in U937 cells did not reveal any genomic difference between it and the virus grown it MOLT-4 cells. The radioimmunoprecipitation analysis of viral proteins showed that the HIV-1-infected U937 cells had a different pattern of envelope glycoproteins and core proteins, which well correlated with the low infectivity of the produced viruses. This experimental system using MOLT-4 and U937 cell lines would be useful to further explore host cell factor(s) which play an important role in the regulation of HIV-1 infectivity.     

Decay Kinetics of Human Immunodeficiency Virus-Specific Effector Cytotoxic T Lymphocytes after Combination Antiretroviral Therapy

Little is known of the changes in human immunodeficiency virus type 1 (HIV-1)-specific effector cytotoxic T lymphocytes (CTL) after potent antiretroviral therapy. Using HLA/peptide tetrameric complexes, we show that after starting treatment, there are early rapid fluctuations in the HIV-1-specific CTL response which last 1 to 2 weeks. These fluctuations are followed by an exponential decay (median half-life, 45 days) of HIV-1-specific CTL which continues while viremia remains undetectable. These data have implications for the immunological control of drug-resistant virus.     

Efficient Gap Repair Catalyzed In Vitro by an Intrinsic DNA Polymerase Activity of Human Immunodeficiency Virus Type 1 Integrase

Cleavage and DNA joining reactions, carried out by human immunodeficiency virus type 1 (HIV-1) integrase, are necessary to effect the covalent insertion of HIV-1 DNA into the host genome. For the integration of HIV-1 DNA into the cellular genome to be completed, short gaps flanking the integrated proviral DNA must be repaired. It has been widely assumed that host cell DNA repair enzymes are involved. Here we report that HIV-1 integrase multimers possess an intrinsic DNA-dependent DNA polymerase activity. The activity was characterized by its dependence on Mg²⁺, resistance to N-ethylmaleimide, and inhibition by 3′-azido-2′,3′-dideoxythymidine-5′-triphosphate, coumermycin A₁, and pyridoxal 5′-phosphate. The enzyme efficiently utilized poly(dA)-oligo(dT) or self-annealing oligonucleotides as a template primer but displayed relatively low activity with gapped calf thymus DNA and no activity with poly(dA) or poly(rA)-oligo(dT). A monoclonal antibody binding specifically to an epitope comprised of amino acids 264 to 273 near the C terminus of HIV-1 integrase severely inhibited the DNA polymerase activity. A deletion of 50 amino acids at the C terminus of integrase drastically altered the gel filtration properties of the DNA polymerase, although the level of activity was unaffected by this mutation. The DNA polymerase efficiently extended a hairpin DNA primer up to 19 nucleotides on a T₂₀ DNA template, although addition of the last nucleotide occurred infrequently or not at all. The ability of integrase to repair gaps in DNA was also investigated. We designed a series of gapped molecules containing a single-stranded region flanked by a duplex U5 viral arm on one side and by a duplex nonviral arm on the other side. Molecules varied structurally depending on the size of the gap (one, two, five, or seven nucleotides), their content of T’s or C’s in the single-stranded region, whether the CA dinucleotide in the viral arm had been replaced with a nonviral sequence, or whether they contained 5′ AC dinucleotides as unpaired tails. The results indicated that the integrase DNA polymerase is specifically designed to repair gaps efficiently and completely, regardless of gap size, base composition, or structural features such as the internal CA dinucleotide or unpaired 5′-terminal AC dinucleotides. When the U5 arm of the gapped DNA substrate was removed, leaving a nongapped DNA template-primer, the integrase DNA polymerase failed to repair the last nucleotide in the DNA template effectively. A post-gap repair reaction did depend on the CA dinucleotide. This secondary reaction was highly regulated. Only two nucleotides beyond the gap were synthesized, and these were complementary to and dependent for their synthesis on the CA dinucleotide. We were also able to identify a specific requirement for the C terminus of integrase in the post-gap repair reaction. The results are consistent with a direct role for a heretofore unsuspected DNA polymerase function of HIV-1 integrase in the repair of short gaps flanking proviral DNA integration intermediates that arise during virus infection.Integration of human immunodeficiency virus type 1 (HIV-1) DNA is an essential step in the replicative cycle of the virus (6, 13, 16, 29, 41). The initial steps whereby HIV-1 DNA becomes covalently associated with the host DNA are mediated by the viral integrase protein. Two distinct chemical reactions are involved. In a processing step, integrase cleaves viral DNA endonucleolytically, resulting in the removal of a GT dinucleotide from the 3′ ends of the DNA (15, 48, 51). Once in the nucleus, concerted cleavage and DNA strand transfer reactions, involving viral and host DNA, enable the processed 3′ termini to become covalently joined to a host DNA target site. The intermediate produced in this manner contains unpaired 5′ ends adjacent to five-base gaps. Completion of integration requires the repair of these gaps and the joining of the 5′ ends of viral DNA to the host DNA (2). The relatively rapid kinetics of 5′-end joining in vivo has been used as a basis on which to argue in favor of a role for integrase in this step of integration (40). Although integrase can catalyze the latter reaction in vitro, albeit inefficiently (28), it has been generally assumed that host cell enzymes perform gap repair and 5′-end joining.Structural, functional, and mutational studies have defined integrase as a 32-kDa protein that can be divided into three distinct functional domains (50). The catalytic core, including amino acids 50 to 212, contains a triad of acidic amino acids (Asp 64, Asp 116, and Glu 152) that form a highly conserved D,D-35-E motif. In the three-dimensional crystal structure, these amino acids are in close proximity (10). Mutation of any one of these acidic residues severely hampers the ability of integrase to catalyze endonucleolytic cleavage and DNA strand transfer (5, 9, 12, 13, 27, 31, 32). The C terminus binds DNA nonspecifically and is required for cleavage and integration activity (47, 49, 52, 53). The amino terminus contains a zinc finger or HHCC motif, which coordinates a molar equivalent of zinc (4). This domain influences DNA binding (21, 25, 47), although it does not bind DNA on its own (26, 38).In the functional integration complex, integrase is believed to act as a multimer (11, 24, 46). Transcomplementation, in which DNA strand transfer and cleavage activities are restored by mixing nonfunctional mutants, implies that the active form of integrase is minimally a dimer (46). Integrase can exist in equilibrium between dimeric and tetrameric forms, and multimerization determinants can be identified within the integrase protein (1). Association of one molar equivalent of zinc with a soluble mutant of integrase favored the formation of the tetrameric form of the protein (54).The present study was undertaken to further characterize HIV-1 integrase by searching for novel enzymatic activities that may be associated with this viral protein. We chose specifically to look for an associated DNA polymerase activity in an attempt to elucidate the final steps in integration, namely, gap repair and 5′-end joining.     

Different In Vivo Effects of HIV-1 Immunodominant Epitope-Specific Cytotoxic T Lymphocytes on Selection of Escape Mutant Viruses

HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.HIV-1-specific cytotoxic T lymphocytes (CTLs) have an important role in the control of HIV-1 replication during acute and chronic phases of an HIV-1 infection (5, 28, 33). On the other hand, HIV-1 can escape from the host immune system by various mechanisms. These may include the appearance of HIV-1 carrying escape mutations in its immunodominant CTL epitopes as well as Nef-mediated downregulation of HLA class I molecules. There is a growing body of evidence for the former mechanism, i.e., that CTLs targeting immunodominant HIV-1 epitopes select escape mutants in chronically HIV-1-infected individuals (18, 20, 36), whereas the latter mechanism was proved by demonstrating that HIV-1-specific CTLs fail to kill Nef-positive-HIV-1-infected CD4⁺ T cells but effectively kill Nef-defective-HIV-1-infected ones or that they suppress the replication of Nef-defective HIV-1 much more than that of Nef-positive HIV-1 (12, 13, 42, 45).It is speculated that HIV-1 immunodominant epitope-specific CTLs have the ability to suppress HIV-1 replication and effectively select escape mutants. However, the correlation between this ability of the CTLs and the appearance of escape mutants is still unclear, because it is not easy to evaluate the ability of HIV-1-specific CTLs to exert a strong immune pressure in vivo. To examine this ability, most previous studies measured the number of HIV-1-specific CTLs or CD8⁺ T cells and the CTL activity against target cells prepulsed with the epitope peptide or those infected with HIV-1 recombinant vaccinia virus (6, 7, 23, 46). However, the results obtained from such experiments do not reflect the ability of the CTLs to exert immune pressure in vivo. We and other groups previously utilized an assay to directly evaluate the ability of the CTLs to suppress HIV-1 replication in vitro (1, 17, 18, 42, 43). This assay may be better for evaluation of immune pressure by HIV-1-specific CTLs than other assays, because the ability of the CTLs to suppress HIV-1 replication is directly measured in cultures of HIV-1-infected CD4⁺ T cells incubated with HIV-1-specific CTL clones. But it still remains unknown whether this assay reflects immune pressure in vivo.In the present study, we investigated whether HIV-1-specific CTLs having a strong ability to suppress HIV-1 replication could positively select escape mutants. Since HLA-A*1101 is known to be an HLA allele relatively associated with a slow progression to AIDS (32), it is speculated that some HLA-A*1101-restricted CTLs would have a strong ability to suppress HIV-1 replication in vitro. Therefore, we first focused on 4 well-known HLA-A*1101-restricted CTL epitopes in the present study. We investigated the frequency of CTLs specific for these epitopes in chronically HIV-1-infected individuals, the ability of these CTLs to suppress HIV-1 replication in vitro, and whether the escape mutants were selected by the CTLs. Furthermore, we analyzed the expression of Programmed Death-1 (PD-1) on these CTLs ex vivo and antigen recognition of them.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

Induction of Potent Human Immunodeficiency Virus Type 1-Specific T-Cell-Restricted Immunity by Genetically Modified Dendritic Cells

A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4(+) and CD8(+) HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.     

Impaired Quality of the Hepatitis B Virus (HBV)-Specific T-Cell Response in Human Immunodeficiency Virus Type 1-HBV Coinfection

Hepatits B virus (HBV)-specific T cells play a key role both in the control of HBV replication and in the pathogenesis of liver disease. Human immunodeficiency virus type 1 (HIV-1) coinfection and the presence or absence of HBV e (precore) antigen (HBeAg) significantly alter the natural history of chronic HBV infection. We examined the HBV-specific T-cell responses in treatment-naïve HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected (n = 24) and HBV-monoinfected (n = 39) Asian patients. Peripheral blood was stimulated with an overlapping peptide library for the whole HBV genome, and tumor necrosis factor alpha and gamma interferon cytokine expression in CD8⁺ T cells was measured by intracellular cytokine staining and flow cytometry. There was no difference in the overall magnitude of the HBV-specific T-cell responses, but the quality of the response was significantly impaired in HIV-1-HBV-coinfected patients compared with monoinfected patients. In coinfected patients, HBV-specific T cells rarely produced more than one cytokine and responded to fewer HBV proteins than in monoinfected patients. Overall, the frequency and quality of the HBV-specific T-cell responses increased with a higher CD4⁺ T-cell count (P = 0.018 and 0.032, respectively). There was no relationship between circulating HBV-specific T cells and liver damage as measured by activity and fibrosis scores, and the HBV-specific T-cell responses were not significantly different in patients with either HBeAg-positive or HBeAg-negative disease. The quality of the HBV-specific T-cell response is impaired in the setting of HIV-1-HBV coinfection and is related to the CD4⁺ T-cell count.There are 40 million people worldwide infected with human immunodeficiency virus type 1 (HIV-1), and 6 to 15% of HIV-1-infected patients are also chronically infected with hepatitis B virus (HBV) (13, 20, 35, 38, 40-42, 47, 50, 61, 69). The highest rates of coinfection with HIV-1 and HBV are in Asia and Africa, where HBV is endemic (33, 68). Following the introduction of highly active antiretroviral therapy (HAART), liver disease is now the major cause of non-AIDS-related deaths in HIV-1-infected patients (12, 13, 38, 59, 65).Coinfection of HBV with HIV-1 alters the natural history of HBV infection. Individuals with HIV-1-HBV coinfection seroconvert from HBV e (precore) antigen (HBeAg) to HBV e antibody less frequently and have higher HBV DNA levels but lower levels of alanine aminotransferase (ALT) and milder necroinflammatory activity on histology than those infected with HBV alone (18, 26, 49). Progression to cirrhosis, however, seems to be more rapid and more common, and liver-related mortality is higher, in HIV-1-HBV coinfection than with either infection alone (47, 59). HBeAg is an accessory protein of HBV and is not required for viral replication or infection; however, chronic HBV infection typically is divided into two distinct phases: HBeAg positive and HBeAg negative (reviewed in reference 15). Most natural history studies of HIV-1-HBV coinfection to date have primarily focused on HBeAg-positive patients from non-Asian countries (23, 44, 46).We previously developed an overlapping peptide library for the HBV genome to detect HBV-specific CD4⁺ and CD8⁺ T-cell responses to all HBV gene products from multiple HBV genotypes (17). In a small cross-sectional study of patients recruited in Australia, we found that in coinfected patients, HBV-specific CD4⁺ T-cell responses, as measured by gamma interferon (IFN-γ) production, were diminished compared to those seen in HBV-monoinfected patients (17). However, patients had varying lengths of exposure to anti-HBV-active HAART at the time of analysis. In this study, therefore, we aimed to characterize the HBV-specific T-cell response in untreated HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected patients and to determine the relationship between the HBV-specific immune response, HBeAg status, and liver disease.     

Distinct Mechanisms Trigger Apoptosis in Human Immunodeficiency Virus Type 1-Infected and in Uninfected Bystander T Lymphocytes

Apoptosis is a main feature of AIDS pathogenesis and is thought to play a role in the progressive decrease of CD4⁺ T lymphocytes in infected individuals. To determine whether apoptosis occurs in infected and/or in uninfected peripheral blood T lymphocytes, we have used a recombinant human immunodeficiency virus type 1 (HIV-1) infectious clone expressing the green fluorescent protein (GFP). Using flow cytometry, we have determined the incidence of apoptosis by either terminal transferase dUTP nick end labeling or annexin-V assays in different cell subpopulations, i.e., in CD4⁺ or CD8⁺ T cells that were GFP positive or negative. After HIV-1 infection of purified peripheral blood lymphocytes, we observed that apoptosis occurred mostly in infected CD4⁺ peripheral blood lymphocytes. Remarkably, the presence of monocyte-derived macrophages in the culture increased dramatically the apoptosis of uninfected bystander T lymphocytes, while apoptosis in HIV-infected T lymphocytes was not changed. We therefore demonstrate that HIV-induced apoptosis results from at least two distinct mechanisms: (i) direct apoptosis in HIV-infected CD4⁺ T lymphocytes and (ii) indirect apoptosis in uninfected T cells mediated by antigen-presenting cells.