Virus-specific cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected chimpanzees.

Chimpanzees have been important in studies of human immunodeficiency virus type 1 (HIV-1) pathogenesis and in evaluation of HIV-1 candidate vaccines. However, little information is available about HIV-1-specific cytotoxic T lymphocytes (CTL) in these animals. In the present study, in vitro stimulation of peripheral blood mononuclear cells (PBMC) from infected chimpanzees with HIV-1 Gag peptides was shown to be a sensitive, reproducible method of expanding HIV-1-specific CD8(+) effector CTL. Of interest, PBMC from two chimpanzees had CTL activity against Gag epitopes also recognized by major histocompatibility complex class I-restricted CTL from HIV-1-infected humans. The use of peptide stimulation will help to clarify the role of CTL in vaccine-mediated protection and HIV-1 disease progression in this important animal model.     

Nef-mediated resistance of human immunodeficiency virus type 1 to antiviral cytotoxic T lymphocytes.

Although Nef has been proposed to effect the escape of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTL) through downmodulation of major histocompatibility complex class I molecules, little direct data have been presented previously to support this hypothesis. By comparing nef-competent and nef-deleted HIV-1 strains in an in vitro coculture system, we demonstrate that the presence of this viral accessory gene leads to impairment of the ability of HIV-1-specific CTL clones to suppress viral replication. Furthermore, inhibition by genetically modified CTL that do not require major histocompatibility complex class I-presented antigen (expressing the CD4 T-cell receptor [TCR] zeta-chain hybrid receptor) is similar for both nef-competent and -deleted strains, indicating that Nef does not impair the effector functions of CTL but acts at the level of TCR triggering. In contrast, we note that another accessory gene, vpr, does not induce resistance of HIV-1 to suppression by CTL clones. We conclude that Nef (and not Vpr) contributes to functional HIV-1 immune evasion and that this effect is mediated by diminished antigen presentation to CTL.     

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.     

Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotoxic T lymphocytes

Telomere length is abnormally short in the CD8(+) T-cell compartment of human immunodeficiency virus type 1 (HIV-1)-infected persons, likely because of chronic cell turnover. Although clonal exhaustion of CD8(+) cytotoxic T lymphocytes (CTL) has been proposed as a mechanism for loss of antigen-specific responses, the functional consequences of exhaustion are poorly understood. Here we used telomerase transduction to evaluate the impact of senescence on CTL effector functions. Constitutive expression of telomerase in an HIV-1-specific CTL clone results in enhanced proliferative capacity, in agreement with prior studies of other human cell types. Whereas the CTL remain phenotypically normal in terms of antigenic specificity and requirements for proliferation, their cytolytic and antiviral capabilities are superior to those of control CTL. In contrast, their ability to produce gamma interferon and RANTES is essentially unchanged. The selective enhancement of cytolytic function in memory CTL by ectopic telomerase expression implies that loss of this function (but not cytokine production) is a specific consequence of replicative senescence. These data suggest a unifying mechanism for the in vivo observations that telomere lengths are shortened in the CD8(+) cells of HIV-1-infected persons and that HIV-1-specific CTL are deficient in perforin. Telomerase transduction could therefore be a tool with which to explore a potential therapeutic approach to an important pathophysiologic process of immune dysfunction in chronic viral infection.     

Ontogeny and specificities of mucosal and blood human immunodeficiency virus type 1-specific CD8(+) cytotoxic T lymphocytes

Induction of adaptive immunity to human immunodeficiency virus type 1 (HIV-1) at the mucosal site of transmission is poorly understood but crucial in devising strategies to control and prevent infection. To gain further understanding of HIV-1-specific T-cell mucosal immunity, we established HIV-1-specific CD8(+) cytotoxic T-lymphocyte (CTL) cell lines and clones from the blood, cervix, rectum, and semen of 12 HIV-1-infected individuals and compared their specificities, cytolytic function, and T-cell receptor (TCR) clonotypes. Blood and mucosal CD8(+) CTL had common HIV-1 epitope specificities and major histocompatibility complex restriction patterns. Moreover, both systemic and mucosal CTL lysed targets with similar efficiency, primarily through the perforin-dependent pathway in in vitro studies. Sequence analysis of the TCRbeta VDJ region revealed in some cases identical HIV-1-specific CTL clones in different compartments in the same HIV-1-infected individual. These results clearly establish that a subset of blood and mucosal HIV-1-specific CTL can have a common origin and can traffic between anatomically distinct compartments. Thus, these effectors can provide immune surveillance at the mucosa, where rapid responses are needed to contain HIV-1 infection.     

Genetic and stochastic influences on the interaction of human immunodeficiency virus type 1 and cytotoxic T lymphocytes in identical twins

Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8+ T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.     

Inhibition of human immunodeficiency virus type 1 replication in primary CD4(+) T lymphocytes, monocytes, and dendritic cells by cytotoxic T lymphocytes

We demonstrate that human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) cytotoxic T lymphocytes (CTL) suppress HIV-1 replication in primary lymphocytes, monocytes, and dendritic cells individually. Viral inhibition is significantly diminished in lymphocyte-dendritic cell clusters, suggesting that these clusters in vivo could be sites where viral replication is more difficult to control by CTL.     

The role of cytotoxic T lymphocytes in human T-cell lymphotropic virus type 1 infection

The vast majority of individuals infected with human T-cell lymphotropic virus type 1 have a strong and persistently activated cytotoxic T lymphocyte response to the virus. Experimental work investigating the effects of human T-cell lymphotropic virus-specific cytotoxic T lymphocytes is conflicting. One significant body of work suggests that specific cytotoxic T lymphocytes are protective and help to reduce the risk of disease. However, another body of work implies that specific cytotoxic T lymphocytes play an important role in the development of disease. Here we use a theoretical model to explore the role of cytotoxic T lymphocytes in persistent infection. A way of reconciling the apparently contradictory data is proposed and experimentally testable predictions are made.     

Non-viral cellular substrates for human immunodeficiency virus type 1 protease

A computer search revealed 10 proteins with homology to the sequence we originally identified in vimentin as the site of cleavage by human immunodeficiency virus type 1 (HIV-1) protease. Of these 10 proteins (actin, alpha-actinin, spectrin, tropomyosins, vinculin, dystrophin, MAP-2, villin, TRK-1 and Ig mu-chain), we show that 4 of the first 5 were cleaved in vitro by this protease, as are MAP-1 and -2 [(1990) J. Gen. Virol. 71, 1985-1991]. In these proteins, cleavage is not restricted to a single motif, but occurs at many sites. However, cleavage is not random, since 9 other proteins including the cytoskeletal proteins filamin and band 4.1 are not cleaved in the in vitro assay. Thus, the ability of HIV-1 protease to cleave specific components of the cytoskeleton may be an important, although as yet unevaluated aspect of the life cycle of this retrovirus and/or may directly contribute to the pathogenesis observed during infection.     

Rat cytotoxic T lymphocytes against human T-lymphotropic virus type 1-infected cells recognize gag gene and env gene encoded antigens

T cell immune responses in syngeneic WKA/H rats were analyzed by using lymphoid cell lines, TARS-1, TART-1, and TARL-2, infected with human T-lymphotropic virus type 1 (HTLV-1). Spleen cells of rats in which these cell lines had been rejected were sensitized in vitro with the same cell lines, and cells cytotoxic to these HTLV-1+ cell lines, and cells cytotoxic to these HTLV-1+ cell lines were generated. The effector cells were CTL of the CD5+ CD8+ phenotype and showed restriction of MHC class I Ag. Direct tests as well as cold target cell inhibition tests with an array of cell populations showed that these CTL reacted only with syngeneic HTLV-1+ cell lines. When xenogeneic HTLV-1+ cell lines were similarly utilized for in vitro sensitization, rat CTL specific for syngeneic HTLV-1+ cells were generated. They were not, however, reactive with xenogeneic HTLV-1+ cells used for sensitization. Syngeneic rat cells selectively expressing gag, env, or pX gene coded Ag were prepared by infection of recombinant vaccinia viruses. In cold target cell inhibition tests of anti-HTLV-1 CTL with thus prepared cells, cytotoxicity against the syngeneic HTLV-1+ cells line, TARS-1, was inhibited by syngeneic cells expressing gag gene or env gene coded Ag. Inhibition was, however, more consistent and more dominant by cells with gag gene than those with env gene. Syngeneic cells with pX gene and MHC class I incompatible cells with gag, env, or pX gene did not inhibit cytotoxicity.     

Fitness landscape of human immunodeficiency virus type 1 protease quasispecies

Here we show, at a high resolution (1%), the human immunodeficiency virus type 1 (HIV-1) protease gene quasispecies landscape from three infected na?ve individuals. A huge range of genetic configurations was found (67%, 71%, and 80% of the nucleotide clones from the three individuals, respectively, were different), and these configurations created a dense net that linked different parts of the viral population. Similarly, a vast diversity of different protease activities was also found. Importantly, 65% of the analyzed enzymes had detectable protease activity, and 11% of the minority individual variants showed similar or better fitness than the master (most abundant) enzyme, suggesting that the viral complexity in this genomic region does not exclusively depend on the enzyme's catalytic efficiency. Several high-fitness minority variants had only one substitution compared to the master sequence, supporting the possibility that the rugged HIV-1 protease quasispecies fitness landscape may be formed by a continuous network that can be traversed by single mutational steps without passing through defective or less-adapted proteins.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize immediate-early protein ICP27.

The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H-2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino-acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity.     

The vpu protein of human immunodeficiency virus type 1 plays a protective role against virus-induced apoptosis in primary CD4(+) T lymphocytes

Previous data revealed that primary cultures of peripheral blood mononuclear cells (PBMCs) were killed by apoptosis at higher rates after infection with two CRF01_AE primary isolates of human immunodeficiency virus type 1 (HIV-1) than after infection with five other CRF01_AE primary isolates, five subtype B primary isolates, and two subtype B laboratory strains. Here, we show evidence that mutations at the vpu gene which were exclusively identified only in the two CRF01_AE isolates mentioned above are involved in their abilities to induce massive apoptosis in primary CD4(+) T lymphocytes. The rates of virus production by these two isolates in the culture media of infected PBMCs were lower (the same as those of the other CRF01_AE isolates) than those of the subtype B isolates. To confirm the correlation between the higher apoptosis-inducing abilities and the mutations at the vpu gene, infectious molecular clone pNL4-3-based vpu mutants were constructed and examined for their apoptosis induction levels. The apoptosis induction levels after introduction of the vpu mutations were greatly increased in primary CD4(+) T lymphocytes. In contrast, the apoptosis induction abilities of these vpu mutants were lower in human T-cell line MT-4. Thus, the Vpu protein of HIV-1 could play a protective role against virus-induced apoptosis in primary CD4(+) T lymphocytes.     

A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8(+) T lymphocytes are discussed.     

Cross-reactive T-helper responses in patients infected with different subtypes of human immunodeficiency virus type 1

Immunization with a recombinant glycoprotein 160 envelope immunogen derived from a virus of genetic subtype B induced strong specific T-helper cell responses in asymptomatic human immunodeficiency virus (HIV) carriers infected with subtypes B to G. This indicates that the HIV-specific T-helper immunity, which is the basis for development of antibodies and cytotoxic T lymphocytes, can be improved by both homologous and heterologous antigens. It also suggests that a particular immunogen can be effective against many different HIV strains.     

Interaction of nuclear protein p140 with human immunodeficiency virus type 1 TAR RNA in mitogen-activated primary human T lymphocytes.

Several lines of evidence suggest that cellular proteins play a role during human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans activation. A recent report from this laboratory has shown that a 140-kDa HeLa nuclear protein (p140) binds specifically to the lower stem region of the Tat response element, TAR RNA. Since HIV-1 trans activation is most efficient in proliferating T cells, we investigated the binding of p140 to TAR RNA in unstimulated and mitogen-activated, G1-phase primary T lymphocytes. TAR RNA/protein-binding activity was low in resting cells but increased significantly within 2 h of activation and remained elevated for at least 48 h. Corresponding increases in p140 protein levels were observed with most but not all donors, suggesting that an additional nuclear factor(s) may be required for efficient binding of this protein to TAR RNA in activated T cells.