Immune recognition of AIDS virus antigens by human and murine cytotoxic T lymphocytes

The CTL response to HIV was analyzed in humans and in mice. By using a novel and strictly autologous lymphocyte culture system, human CTL lines were established with PBL from seropositive asymptomatic donors and from patients suffering from AIDS or presenting AIDS-related complex. CTL from HLA-A2 donors recognize and kill murine P815 mastocytoma cells doubly transfected with the human HLA-A2 gene and the HIV env gene; they also kill HLA-compatible human macrophages infected with HIV. CTL specific for the HIV env Ag were also generated in BALB/c mice by immunization with syngeneic murine cells transfected with the HIV env gene. Human and murine HIV-immune CTL populations belong to the CD8 subset of T lymphocytes and are restricted by class I HLA or H-2 transplantation Ag, respectively, in the recognition of HIV env Ag. The two different experimental systems presented here can be used to study CD8 lymphocyte immunity against HIV. The murine model of CTL immunity offers the additional advantage of avoiding the manipulation of infectious virus isolates.     

HIV infection in India: Epidemiology, molecular epidemiology and pathogenesis

The year 1986 saw first case of HIV infection as well as first report of AIDS case in India. Since then the epidemic has spread throughout the country. In the recent years there is evidence of epidemic being stabilized with decrease in new infections reported from some parts of the country. The absolute number of HIV infections in the country is expected to be close to 2.5 million and National AIDS Control Programme, phase III is geared to contain the epidemic. HIV viruses circulating in India predominantly belong to HIV-1 subtype C. However, there have been occasional reports of HIV-1 subtype A and B. Matter of concern is reports of A/C and B/C mosaic viruses that are being reported from different parts of the country. The data on HIV drug resistance from India is rather limited. Most of the studies have shown that the virus strains from drug naïve patients do not show significant level of drug resistance mutations. The few immunological studies in Indian patients show that the Indian HIV infected patients show both HIV-specific CTL responses as well as neutralizing antibody response. Mapping of CTL epitopes showed that while Indian patients identify same regions of Gag antigen as recognized by South African subtype C infected patients, some regions are uniquely recognized by Indian patients. There are very few studies on host genetic factors in India in context with HIV infection. However there are evidences reported of association of host genetic factors such as HLA types and haplotypes and HIV disease.     

Epitope recognition of conserved HIV envelope sequences by human cytotoxic T lymphocytes.

CTL constitute an essential part of the immune response against the HIV. CTL recognize peptides derived from viral proteins together with the MHC class I molecules on the surface of infected cells. The CTL response could be important in prevention or control of infection with HIV by destroying virus-producing cells. In this study we have attempted to identify peptide epitopes recognized by HIV-specific CTL. Using synthetic peptides, we have identified six conserved peptidic epitopes on the gp120 envelope glycoprotein recognized by polyclonal human CTL in association with HLA-A2 class I transplantation Ag. These results were confirmed by two approaches: i) blocking of CTL activities with antibodies specific for three of these conserved peptides; and ii) construction of doubly transfected P815-A2 target cells, using deletions of the HIV env gene. Vaccination or immunotherapy in HLA-A2 individuals can thus be considered using highly conserved HIV env peptidic sequences.     

Circumvention of defective CD4 T helper cell function in HIV-infected individuals by stimulation with HLA alloantigens

PBL from approximately 50% of asymptomatic individuals infected with HIV have been previously demonstrated to exhibit defective in vitro Th function that is selective for influenza A virus (FLU), but not for HLA alloantigens (ALLO). In this report, we have further studied HIV+ individuals with this selective Th defect, and demonstrate that defective in vitro CTL responses to FLU can be restored by costimulation with FLU + ALLO. In contrast, HIV+ patients who have lost Th responses to ALLO were unable to correct CTL responses to FLU by this costimulation procedure. These findings indicate that intact Th responses to ALLO can be used in vitro to provide Th signals necessary to activate the T effector cell response to a potential pathogenic virus. Our results raise the possibility that a program of in vivo coimmunization with ALLO plus antigens of potential pathogens (including HIV) can be useful in HIV+ patients exhibiting selective defects in Th function. Furthermore, this approach could be incorporated in vaccine trials aimed at enhancing immunity to HIV in patients who have been infected previously with this virus.     

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.     

A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6.

A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli. The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides. The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced. Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone.     

MULTIPLE HIV-1 INFECTION OF CELLS AND THE EVOLUTIONARY DYNAMICS OF CYTOTOXIC T LYMPHOCYTE ESCAPE MUTANTS

Cytotoxic T lymphocytes (CTL) are an important branch of the immune system, killing virus-infected cells. Many viruses can mutate so that infected cells are not killed by CTL anymore. This escape can contribute to virus persistence and disease. A prominent example is HIV-1. The evolutionary dynamics of CTL escape mutants in vivo have been studied experimentally and mathematically, assuming that a cell can only be infected with one HIV particle at a time. However, according to data, multiple virus particles frequently infect the same cell, a process called coinfection. Here, we study the evolutionary dynamics of CTL escape mutants in the context of coinfection. A mathematical model suggests that an intermediate strength of the CTL response against the wild-type is most detrimental for an escape mutant, minimizing overall virus load and even leading to its extinction. A weaker or, paradoxically, stronger CTL response against the wild-type both lead to the persistence of the escape mutant and higher virus load. It is hypothesized that an intermediate strength of the CTL response, and thus the suboptimal virus suppression observed in HIV-1 infection, might be adaptive to minimize the impact of existing CTL escape mutants on overall virus load.     

Recognition of viral antigens by human influenza A virus-specific T lymphocyte clones

The nature of the viral antigens recognized by influenza A virus-immune cytotoxic T lymphocytes (CTL) is still a matter of debate. We have used four human influenza A virus-specific T lymphocyte clones with antigen-specific cytotoxic and proliferative activity to investigate the requirements for recognition of viral antigens on infected cells. One clone recognized a cross-reactive determinant on the viral hemagglutinin, and two clones were specific for different epitopes on the viral nucleoprotein (NP). A fourth clone seemed to be specific for the viral M protein. Target cell recognition was abrogated by the addition, during infection, of the lysosomotropic drug chloroquine, known to inhibit antigen processing. Furthermore, target cells that had been pulsed with soluble purified NP were recognized and were lysed by the NP-specific clone. This reaction could also be abrogated by the addition of chloroquine during pulsing. These results were obtained irrespective of whether EBV-transformed B lymphoblastoid cells or Ia antigen-expressing T cell blasts were used as target cells. It is concluded that CTL can recognize internal viral proteins that are actively presented at the surface of the target cell. These data indicate that probably every viral protein can function as a target molecule for virus-immune CTL.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor

HIV's considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A(*)02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (K(D) < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.     

The role of HIV replicative fitness in perinatal transmission of HIV

Perinatal transmission of Human immunodeficiency virus(HIV),also called mother-to-child transmission(MTCT),accounts for 90% of infections in infants worldwide and occurs in 30%-45% of children born to untreated HIV-1 infected mothers.Among HIV-1 infected mothers,some viruses are transmitted from mothers to their infants while others are not.The relationship between virologic properties and the pathogenesis caused by HIV-1 remains unclear.Previous studies have demonstrated that one obvious source of selective pressure in the perinatal transmission of HIV-1 is maternal neutralizing antibodies.Recent studies have shown that viruses which are successfully transmitted to the child have growth advantages over those not transmitted,when those two viruses are grown together.Furthermore,the higher fitness is determined by the gp120 protein of the virus envelope.This suggests that the selective transmission of viruses with higher fitness occurred exclusively,regardless of transmission routes.There are many factors contributing to the selective transmission and HIV replicative fitness is an important one that should not be neglected.This review summarizes current knowledge of the role of HIV replicative fitness in HIV MTCT transmission and the determinants of viral fitness upon MTCT.     

＂Unconventional＂ Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However,only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes,such as high sequence diversity,heavy glycosylation,and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohe-magglutinin (PHA)-stimulated human PBMCs as target cells. Thus,in essence the assay evaluates HIV-1 replication in CD4 T cells. Recently,several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera,although they do not have neutralizing activity when tested by the "conventional" neutralization assay,do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications,through opsonization of virus particles into macrophages and immature dendritic cells (iDCs),or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

"Unconventional" Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

Analysis of the human env-specific cytotoxic T-lymphocyte (CTL) response in natural human immunodeficiency virus type 1 infection: low prevalence of broadly cross-reactive env-specific CTL.

Major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to persistent virus infections. Candidate vaccines against human immunodeficiency virus type 1 (HIV-1) should elicit broad cross-reactive immunity to confer protection against different strains of HIV-1. As it is likely that candidate vaccines will include the envelope gene product Env, we determined the proportion of CTL clones which recognized variable and conserved determinants in three env variants during natural infection. Limiting dilution analysis was used to characterize numerous short-term CTL clones derived from peripheral blood of HIV-1-infected subjects, using split-well analysis to assay cytotoxicity against target cells expressing gp160env of HIV-1 strains IIIB, MN, and RF. In 9 of 12 HIV-1-infected subjects, at the clonal level most env-specific CTL recognized determinant(s) within one env variant but not in the other variants. In some subjects, CTL recognized multiple nonconserved determinants in different variants. The pattern of recognition of different env variants was relatively stable over time. In most of the patients studied, the proportion of CTL which showed cross-recognition of conserved determinants shared among the three strains was low. Two novel CTL epitopes within gp41 were identified by using 15-mer peptides of the HIV-SF2 sequence. When specific peptide was used to stimulate CTL precursors in vitro, the frequency of peptide-specific CTL precursors was very high, but the CTL elicited by this stimulation were highly strain specific. We conclude that the use of a single HIV env variant to detect CTL activity can underestimate the magnitude and complexity of the env-specific CTL response. The low prevalence of CTL clones which show cross-recognition of conserved determinants may have implications for immunization strategies based solely on env; to elicit broadly cross-reactive CTL other, more conserved viral antigens are likely to be needed in addition to env. Because of its capacity to distinguish CTL responses against different virus strains, limiting dilution analysis is particularly appropriate to quantitate the immune responses generated by candidate env-based vaccines.     

Infectious HIV-1 assembles in late endosomes in primary macrophages

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.     

Unique CRF01_AE Gag CTL epitopes associated with lower HIV-viral load and delayed disease progression in a cohort of HIV-infected Thais

Cytotoxic T Lymphocytes (CTLs) play a central role in controlling HIV-replication. Although numerous CTL epitopes have been described, most are in subtype B or C infection. Little is known about CTL responses in CRF01_AE infection. Gag CTL responses were investigated in a cohort of 137 treatment-naïve HIV-1 infected Thai patients with high CD4+ T cell counts, using gIFN Enzyme-Linked Immunospot (ELISpot) assays with 15-mer overlapping peptides (OLPs) derived from locally dominant CRF01_AE Gag sequences. 44 OLPs were recognized in 112 (81.8%) individuals. Both the breadth and magnitude of the CTL response, particularly against the p24 region, positively correlated with CD4+ T cell count and inversely correlated with HIV viral load. The breadth of OLP response was also associated with slower progression to antiretroviral therapy initiation. Statistical analysis and single peptide ELISpot assay identified at least 17 significant associations between reactive OLP and HLA in 12 OLP regions; 6 OLP-HLA associations (35.3%) were not compatible with previously reported CTL epitopes, suggesting that these contained new CTL Gag epitopes. A substantial proportion of CTL epitopes in CRF01_AE infection differ from subtype B or C. However, the pattern of protective CTL responses is similar; Gag CTL responses, particularly against p24, control viral replication and slow clinical progression.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

Dynamic interplay between viral adaptation and immune recognition during HIV-1 infection

Untreated human immunodeficiency virus (HIV) infections usually lead to death from AIDS, although the rate of the disease progression varies widely among individuals. The cytotoxic T lymphocyte (CTL) response, which is restricted by highly polymorphic MHC class I alleles, plays a central role in controlling HIV replication. It is now recognized that the antiviral efficacy of CTLs at the single cell level is dependent on their antigen specificity and is important in determining the quality of host response to viruses so that the individual will remain asymptomatic. However, because of the extreme mutational plasticity of HIV, HIV-specific CTL responses are continuously and dynamically changing. In order to rationally design an effective vaccine, the questions as to what constitutes an effective antiviral CTL response and what characterizes a potent antigenic peptide to induce such responses are becoming highlighted as needing to be answered.