Multiple levels of PKR inhibition during HIV-1 replication

Recent therapeutic approaches against HIV-1 include IFN in combination therapy for patients with coinfections or as an alternative strategy against the virus. These treatment options require a better understanding of the weak efficacy of the IFN-stimulated genes, such as the protein kinase RNA-activated (PKR), which results in viral progression. Activated PKR has a strong antiviral activity on HIV-1 expression and production in cell culture. However, PKR is not activated upon HIV-1 infection when the virus reaches high levels of replication, due to viral and cellular controls. PKR is activated by low levels of the HIV-1 trans-activation response (TAR) RNA element, but is inhibited by high levels of this double-stranded RNA. The viral Tat protein also counteracts PKR activation by several mechanisms. In addition, HIV-1 replicates only in cells that have a high level of the TAR RNA binding protein (TRBP), a strong inhibitor of PKR activation. Furthermore, increased levels of adenosine deaminase acting on RNA (ADAR1) are observed when HIV-1 replicates at high levels and the protein binds to PKR and inhibits its activation. Finally, the PKR activator (PACT) also binds to PKR during HIV-1 replication with no subsequent kinase activation. The combination of all the inhibiting pathways that prevent PKR phosphorylation contributes to a high HIV-1 production in permissive cells. Enhancing PKR activation by counteracting its inhibitory partners could establish an increased innate immune antiviral pathway against HIV-1 and could enhance the efficacy of the IFN treatment.     

Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1

Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.     

Efficient expansion of HIV-1-specific T cell responses by homologous immunization with recombinant Semliki Forest virus particles

Vaccines based on recombinant viruses represent a promising strategy for the development of a prophylactic vaccine against HIV-1. However, despite a proven capacity to stimulate potent HIV-1-specific immune responses, viral systems have limited utility in homologous prime-boost regimens due to the generation of anti-vector immune responses. It is therefore important to develop a diverse set of vaccine candidates that can be combined in different heterologous prime-boost regimens and/or to identify a vaccine candidate that is less sensitive to anti-vector mediated immunity. In this report, we describe the design and pre-clinical immunogenicity of a Semliki Forest virus-based vaccine, VREP-C, encoding Indian origin HIV-1 clade C antigens. We show that a single immunization with VREP-C stimulates HIV-1-specific IFNgamma ELISPOT responses, which were efficiently boosted by a second and a third homologous VREP-C immunization resulting in highly potent cytotoxic T cell responses. These results suggest that VREP-C may be a valuable component of a future prophylactic vaccine against HIV-1.     

Multiepitope vaccines intensively increased levels of antibodies recognizing three neutralizing epitopes on human immunodeficiency virus-1 envelope protein

A few neutralizing antibodies against human immunodeficiency virus-1 (HIV-1) envelope proteins have been shown to be highly effective at neutralizing different strains in vitro, and exist at very low levels in the sera of HIV-1-infected individuals. Based on our hypothesis that epitope vaccination may be a novel strategy for inducing high levels of antibodies against HIV-1, we prepared multiepitope vaccines using three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope proteins. The PI [C-G-(ELDKWA-GPGRAFY)2-K] and PII (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) peptides were synthesized and conjugated to a carrier protein, bovine serum albumin (BSA). After vaccination, both the PI-BSA and PII-BSA multiepitope vaccines induced high levels of epitope-specific antibodies to the three neutralizing epitopes (antibody titre: 1 : 12,800-102,400). The recombinant glycoprotein 160 (rgp160) subunit vaccine induced strong antibody responses to rgp160, but only very weak epitope-specific antibody responses to the three epitopes. The epitope-specific antibodies were isolated from rabbit sera by single epitope-peptide-conjugated sepharose columns. A yield of 51 microg of epitope-specific antibodies/ml of serum (mean value) was obtained and identified to recognize these epitopes, while 0.35 microg of protein was isolated from 1 ml of pooled preserum by C-(ELDKWAG)4- or C-(RILAVERYLKD-G)2-K- and C-(GPGRAFY)4-sepharose columns. The levels of these epitope-specific antibodies induced in rabbits were much greater than 1 microg/ml, a level that is considered to confer long-term protection against some viruses. Moreover, these antibodies recognized the neutralizing epitopes on peptides and rgp41. Based on the fact that a very low level of ELDKWA epitope-specific antibodies exist in HIV-1-infected individuals, these results suggesting that synthetic epitope vaccines could induce high levels of multiepitope-specific neutralizing antibodies indicate a new strategy for developing an effective neutralizing antibody-based epitope/peptide vaccine against HIV-1.     

CD8+ T cell responses to human immunodeficiency viruses type 2 (HIV-2) and type 1 (HIV-1) gag proteins are distinguishable by magnitude and breadth but not cellular phenotype

The mechanisms underlying the relatively slow progression of human immunodeficiency virus type 2 (HIV-2) compared with HIV-1 infection are undefined and could be a result of more effective immune responses. We used HIV-2 and HIV-1 IFN-gamma enzyme-linked immunospot assays to evaluate CD8(+) T cell responses in antiretroviral-naive HIV-2- ('HIV-2(+)') and HIV-1-infected ('HIV-1(+)') individuals. Gag-specific responses were detected in the majority of HIV-2(+) and HIV-1(+) subjects. Overlapping gag peptide analysis indicated a significantly greater magnitude and breadth of responses in the HIV-1(+) cohort, and this difference was attributable to low responses in HIV-2(+) subjects with undetectable viral load (medians 2107 and 512 spot-forming units per 10(6) PBMC, respectively, p=0.007). We investigated the phenotype of viral epitope-specific CD8(+) T cells identified with HLA-B53- and HLA-B58-peptide tetramers (8 HIV-2(+), 11 HIV-1(+) subjects). HIV-2-specific CD8(+) T cells were predominantly CD27(+) CD45RA(-), and only a minority expressed perforin. The limited breadth and low frequency of CD8(+) T cell responses to HIV-2 gag in aviremic HIV-2(+) subjects suggests that these responses reflect antigen load in plasma, as is the case in HIV-1 infection. Immune control of HIV-2 does not appear to be related to the frequency of perforin-expressing virus-specific CD8(+) T cells.     

Antigenicity and Predefined Specificities of the Multi-Epitope Vaccine in Candidate Consisting of Neutralizing Epitope and Mutated Epitopes Suggested a New Way against HIV-1 Mutation

A seven-amino acid epitope GPGRAFY located inside the V3 loop on envelope protein gp120 of HIV-1 is the principal neutralizing epitope (PNE), and a subset of anti-V3 antibodies specific for this epitope show a broad range of neutralizing activity. But this epitope undergoes restricted mutation. In this study, three epitope peptides [C-(GPGRAFY)2, C-(GPGQTFY)2 and C-(GPGQAWY)2] that contain neutralizing epitope GPGRAFY and its two mutated epitope GPGQTFY and GPGQAWY, were synthesized and then conjugated to carrier protein KLH (keyhole limpet hemocyanin). the epitope-vaccines C-(GPGRAFY)2-KLH, C-(GPGQTFY)2-KLH and C-(GPGQAWY)2-KLH induced high levels of antibodies to three V3 loop peptides that contain these epitopes respectively, and the antibody response induced by each epitope-vaccine showed predefined epitope-specific. When these three epitope-peptides mixed together and conjugated to carrier protein, or conjugated to carrier protein separately and then mixed together, high levels of epitope-specific antibodies which respectively recognized these epitopes on V3 loop peptide and both mutated peptides all can be induced by both of them. In blotting assay, these epitope-specific antibodies all recognized the neutralizing epitope and mutated epitopes on peptides respectively. In addition, the reactivity of the antibodies with whole gp120 molecule which contained the epitope GPGRAFY was tested. Only the GPGRAFY-epitope-specific antibodies but not the other antibodies recognized the gp120 molecule. These results provide experimental evidence that the candidate multi-epitope-vaccine containing neutralizing epitope and mutated epitopes may bring new hope against viral mutation resulting in HIV-1 immune evasion and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1 infection.     

Manipulating both the inhibitory and stimulatory immune system towards the success of therapeutic vaccination against chronic viral infections

Summary: One potentially promising strategy to control chronic infections such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus is therapeutic vaccination, which aims to reduce persisting virus by stimulating a patient's own antiviral immune responses. However, this approach has fallen short of expectations, because antiviral T cells generated during chronic infections often become functionally exhausted and thus do not respond properly to therapeutic vaccination. Therefore, it is necessary to develop a therapeutic vaccine strategy to more effectively boost endogenous T-cell responses to control persistent viral infections. Studies to elucidate the cause of impaired T-cell function have pointed to sustained inhibitory receptor signaling through T-cell expression of programmed death 1 (PD-1). Recently, another inhibitory molecule, cytotoxic T lymphocyte antigen 4 (CTLA-4), and also an immunosuppressive cytokine, interleukin 10 (IL-10), have been reported to be potential factors of establishing immune suppression and viral persistence. Blocking these negative signaling pathways could restore the host immune system, enabling it to respond to further stimulation. Indeed, combining therapeutic vaccination along with the blockade of inhibitory signals could synergistically enhance functional CD8⁺ T-cell responses and improve viral control in chronically infected mice, providing a promising strategy for the treatment of chronic viral infections. Furthermore, not only the ablation of negative signals but also the addition of stimulatory signals, such as interleukin 2 (IL-2), might prove to be a potentially promising strategy to augment the efficacy of therapeutic vaccination against chronic viral infections.     

Rapid evolution of HIV-1 to functional CD8⁺ T cell responses in humanized BLT mice

The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1-specific immune responses and viral evolution during the acute phase of infection. HIV-1-specific CD8(+) T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8(+) T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen-specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.     

Induction of dendritic cell-mediated immune responses against HIV-1 by antigen-capturing nanospheres in mice

Prophylactic vaccines, designed to elicit potent humoral and cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens in mucosa, are the important approach to the protection of individuals against HIV-1 infection, since HIV-1 transmission is largely a result of sexual contact. In this study, a novel strategy has been developed to induce HIV-1-specific immune responses, which involves inactivated HIV-1-caputring concanavalin A (Con A)-immobilized nanospheres (HIV-NS) and their interaction with bone marrow (BM)-derived dendritic cells. HIV-NS were taken up by dendritic cells via cytoskeleton-dependent but mannose-binding site-independent phagocytosis. Serial stimulations to unprimed T-cells with HIV-1 gp120-capturing NS-pulsed dendritic cells could induce antigen-specific T-cell response. Intranasal administration of fluorescein isothiocyanate-labeled nanospheres (NS) in mice proved that the particles were taken up into pulmonary dendritic cells. Analysis of mice receiving intranasal immunizations with HIV-NS revealed that the mice efficiently induced the antibodies against HIV-1 in the genital tract and specific cytotoxic T-cells in the spleen. These results suggest that the use of HIV-1-NS may provide a novel and promising approach for the induction of humoral and cellular immune responses to HIV-1.     

Innate immunity and chronic immune activation in HCV/HIV-1 co-infection

Innate immune responses are critical in the defense against viral infections. NK cells, myeloid and plasmacytoid dendritic cells, and invariant CD1d-restricted NKT cells mediate both effector and regulatory functions in this early immune response. In chronic uncontrolled viral infections such as HCV and HIV-1, these essential immune functions are compromised and can become a double edged sword contributing to the immunopathogenesis of viral disease. In particular, recent findings indicate that innate immune responses play a central role in the chronic immune activation which is a primary driver of HIV-1 disease progression. HCV/HIV-1 co-infection is affecting millions of people and is associated with faster viral disease progression. Here, we review the role of innate immunity and chronic immune activation in HCV and HIV-1 infection, and discuss how mechanisms of innate immunity may influence protection as well as immunopathogenesis in the HCV/HIV-1 co-infected human host.     

Modeling immune intervention strategies for HIV-1 infection of humans in the macaque model

The introduction of potent antiretroviral therapy (ART) has generated hope and prospects in the management of human immunodeficiency virus-1 (HIV-1) infection. Long-term side effects of ART, however, have also indicated the limitations of this approach alone. A decade ago, immune therapy trials in HIV-1-infected individuals were performed in the absence of ART with a gp160-based vaccine [1]. At that time, the notion that the HIV-1 env obtained from lab-adapted strains would be a poor inducer of antibodies able to neutralize primary HIV-1 isolates was in its infancy [2]. Similarly, the importance of cell-mediated immune response in the containment of HIV-1 replication both in acute and chronic infection [3], [4], [5], [6], [7] and [8], albeit predictable from other models of viral infection, was not fully appreciated. Here, attempts to model immune intervention in SIVmac251-infected macaques using vaccines able to elicit cell-mediated immune responses will be reviewed.The introduction of ART has resulted in effective suppression of viral replication and decreased morbidity and mortality of HIV-1-infected individuals [9]. The decreased morbidity appears to be associated with the reconstitution of immune responses to pathogens, such as cytomegalovirus and Epstein-Barr virus [10], [11] and [12]. However, HIV-1-specific immune responses decline during ART treatment of adults and children [13], [14], [15], [16] and [17], perhaps because of a decrease of HIV-1 replication and antigen exposure under ART. The wide use of ART has also resulted in a better appreciation of the limitations of this daily multi-drug combination treatment, such as compliance [18] and [19], due to the complexity of treatment and life-threatening side effects after long-term treatment [20], [21], [22], [23], [24], [25], [26], [27], [28], [29] and [30].     

Progress on the Induction of Neutralizing Antibodies Against HIV Type 1 (HIV-1)

Infection with HIV type 1 (HIV-1), the causative agent of AIDS, is one of the most catastrophic pandemics to affect human healthcare in the latter 20th century. The best hope of controlling this pandemic is the development of a successful prophylactic vaccine. However, to date, this goal has proven to be exceptionally elusive. The recent failure of an experimental vaccine in a phase IIb study, named the STEP trial, intended solely to elicit cell-mediated immune responses against HIV-1, has highlighted the need for a balanced immune response consisting of not only cellular immunity but also a broad and potent humoral antibody response that can prevent infection with HIV-1. This article reviews the efforts made up to this point to elicit such antibody responses, especially with regard to the use of a DNA prime-protein boost regimen, which has been proven to be a highly effective platform for the induction of neutralizing antibodies in both animal and early-phase human studies.     

Differential cytotoxic T-lymphocyte (CTL) responses in HIV-1 immunised sibling chimpanzees with shared MHC haplotypes

Cell mediated immune responses to HIV-1 and CTL responses in particular differ dramatically in infected individuals. This may largely be influenced by the immunogenetic differences of different individuals such as those encoded by the MHC. These differences may be difficult to dissect due to the immunosuppressive nature of HIV-1 infection itself. In order to reduce the variables associated with effects of the virus, one recombinant viral antigen was chosen from a particular HIV-1 variant (rgp120 of the clinical isolate HIV-1w6.1D). To minimise differences between outbred hosts, we chose two sibling chimpanzees from which the family pedigree and genetic segregation with respect to polymorphic MHC molecules was known. Immunisation induced strong antigen specific antibody and T-helper immune responses. The magnitude and persistence of the humoral and T-helper immune responses were comparable in both chimpanzees. However, CTL responses were only observed in one sibling. These responses were subsequently mapped to several distinct epitopes. The CTL response to the immunodominant epitope was found to be presented in the context of a MHC molecule which was shared by both siblings. The absence of a CTL response in the other sibling is not yet understood, but could not be attributed to MHC alleles that were not shared by these two chimpanzees. These findings suggest that other polymorphic immunoregulatory mechanisms such as those involved in antigen processing and presentation influence host CTL responses to HIV-1.     

Chimeric immune receptor T cells bypass class I requirements and recognize multiple cell types relevant in HIV-1 infection

Transduction of T cells with a chimeric immune T cell receptor (CIR) has been proposed as a strategy to generate cellular immunity against viral pathogens such as HIV-1. In the case of the CD4-CD3-zeta chain (CD4-zeta) CIR, specificity for HIV-1 is conferred by binding of the CD4 moiety to gp120 on the surface of infected cells. However, it is unclear whether CD4-zeta-T cells may differ from naturally derived CD8(+) cytotoxic T cells (CTL) in their susceptibility to viral escape mechanisms or ability to recognize different cell types that support viral replication. We demonstrate that CIR-T cells can mediate antiviral activity against HIV-1 in cells that are resistant to class I-restricted CTL-mediated activity. Furthermore, CIR-T cells can suppress virus in multiple cell types, including monocytes, dendritic cells, and lymphocyte-dendritic cell clusters. These results provide evidence that T cells can be redirected against novel targets, and that independence from the class I pathway may have distinct advantages.     

ELNKWA-epitope specific antibodies induced by epitope-vaccine recognize ELDKWA- and other two neutralizing-resistant mutated epitopes on HIV-1 gp41

Based on the fact that monoclonal antibody (mAb) 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 separately or in combination with other mAbs showed potent neutralizing activity to a wide range of primary HIV-1 isolates in vivo and in vitro, but this epitope undergoes restricted mutation. ELNKWA is a neutralizing-resistant mutated epitope. We induced ELNKWA-epitope-specific polyclonal and monoclonal antibodies and studied the interaction of the antibodies with ELDKWA-epitope and other two neutralizing-resistant mutated epitopes. The candidate ELNKWA-epitope-vaccine induced a high level of antibodies to the ELNKWA-epitope-peptide. The ELNKWA-epitope-specific polyclonal antibodies bound not only the ELNKWA-, but also ELDKWA-, ELEKWA- and ELDEWA-epitope-peptides in ELISA-assay. Moreover, the antibodies also recognized four C-domain-peptides (P5, P6, P7, P8) which contain these four epitopes, respectively. Interestingly, an ELNKWA-epitope-specific monoclonal antibody (TH-Ab1) induced by the candidate ELNKWA-epitope-vaccine could also recognize the four C-domain-peptides containing ELNKWA-, ELDKWA-, ELEKWA- and ELDEWK-epitopes. These results indicate that the candidate ELNKWA-epitope-vaccine could induce high levels of antibodies, which recognize the neutralizing epitope ELDKWA and three neutralizing-resistant mutated epitopes, suggesting that the candidate ELNKWA-epitope-vaccine may help to overcome the problem of viral escape from neutralization through mutation at D or K position, and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1.     

Las células natural killer y su papel en la respuesta inmunitaria durante la infección por el virus de la inmunodeficiencia humana tipo-1

During human immunodeficiency virus type-1 (HIV-1) infection there are several changes in the frequency, phenotype and function of NK cells, altering their antiviral response. This is correlated with increased viral loads, and AIDS progression. However, studies in individuals with natural resistance to HIV-1 infection have shown that NK cells are very important in controlling viral replication, not only for their antiviral activity, but also because of their effects on the activity of other innate immune cells, such as dendritic cells. NK cells do not have antigen receptors, but it has been recently reported that they can specifically respond to HIV-1 peptides. Although the mechanism is not fully elucidated, this finding open more options in the study of new therapeutic or preventative strategies against HIV-1 infection.     

MCMV-based vaccine vectors expressing full-length viral proteins provide long-term humoral immune protection upon a single-shot vaccination

Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a β-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8⁺ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMV^HA) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMV^S). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMV^HA-vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to the effects of memory T cells. Conclusively, we show here that MCMV vectors induce not only long-term cellular immunity but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens.     

Design and construction of a recombinant epitope-peptide gene as a universal epitope-vaccine strategy

The multiple sequence and antigenic variability of HIV-1 has led to unprecedented difficulties in the development of effective vaccines. More and more experimental evidence suggests that HIV-1 mutants have resulted in immune evasion and pose a challenge for conventional efforts to prepare effective vaccines. We have suggested that an epitope-vaccine could offer a new strategy against HIV-1. Several candidate epitope-vaccines were prepared and systematically studied in animal experiments. Here, we have proposed a universal method for the preparation of immunogens for an epitope-vaccine by genetic engineering. With this method, copies of epitopes from one or more antigens can be conveniently combined into one recombinant immunogen, which may also help to design epitope-vaccines against other viral infections.     

Induction of HIV-1-specific T cell responses by administration of cytokines in late-stage patients receiving highly active anti-retroviral therapy.

Highly active anti-retroviral therapy (HAART) is associated with reduction in the morbidity and mortality of patients with advanced HIV-1 disease. The ability of such treatment to improve immune responses against HIV-1 and opportunistic pathogens is variable and limited. Addition of cytokine immunotherapy to this treatment may improve immune responses. IL-2 with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered to HIV-1+ individuals receiving HAART with undetectable viral loads, and CD4 counts < 100 cells/microl. In one patient presenting with Mycobacterium avium complex (MAC) infection, we evaluated the effect of cytokine immunotherapy on lymphocyte phenotype; plasma viral load; proliferative responses to mitogens, recall and HIV-1 antigens; cytokine production and message in response to non-specific and specific stimuli; and natural killer (NK) cell activity. Proliferation assays were performed in two similar patients. Before cytokine immunotherapy the predominant CD8+ population was mainly CD28-. No proliferation or IL-2 production was seen in response to mitogens, recall or HIV-1 antigens; and no HIV-1 peptide-specific interferon-gamma (IFN-gamma)-secreting cells were present. Low levels of IL-4 were detected in response to antigens to which patients had been exposed, associated with up-regulated expression of costimulatory molecules influenced by IL-4. Following IL-2 administration, loss of IL-4 was associated with increased NK cell activity and HIV-1 peptide-specific and non-specific IFN-gamma-producing cells. Proliferative responses associated with IL-2 production and responsiveness were only seen after subsequent concomitant administration of GM-CSF with IL-2. These changes mirrored clinical improvement. An imbalance of lymphocyte subsets may account for immune unresponsiveness when receiving HAART. Restoration of responses following immunotherapy suggests a shift towards a lymphocyte profile with anti-pathogen activity.     

Induction of single and dual cytotoxic T-lymphocyte responses to viral proteins in mice using recombinant hybrid Ty-virus-like particles.

The induction of cytotoxic T-lymphocyte (CTL) responses to viral proteins is thought to be an essential component of protective immunity against viral infections. Methods for generating such responses in a reproducible manner would be of great value in vaccine development. We demonstrate here that the recombinant antigen-presentation system based on the yeast transposon (Ty) particle-forming p1 protein is a potent means of inducing CTL responses to a variety of viral CTL epitopes, including influenza virus nucleoprotein (two epitopes), Sendai virus and vesicular stomatitis virus nucleoproteins, and the V3 loop of human immunodeficiency virus type-1 (HIV-1) gp120. CTL were primed by hybrid Ty-virus-like particles (VLP) carrying the minimal epitope or as much as 19,000 MW of protein. Ty-VLP carrying two different epitopes (dual-epitope Ty-VLP) were capable of priming CTL responses in two different strains of mice or against two epitopes in the same individual. Furthermore, co-administration of a mixture of two different Ty-VLP carrying single epitopes could induce responses to both epitopes in the same individual. Ty-VLP appear to represent a reproducible and flexible system for inducing CTL responses in mice, and warrant further evaluation in primates.