Major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes from horses with equine infectious anemia virus recognize Env and Gag/PR proteins.

Cytotoxic T lymphocytes (CTL) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. Since there is limited evidence for an in vivo role of CTL in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. Horses infected with equine infectious anemia virus (EIAV) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. Viremic episodes may recur, but most horses ultimately control infection and become asymptomatic carriers. To begin dissection of the immune mechanisms involved in EIAV control, peripheral blood mononuclear cells (PBMC) from infected horses were evaluated for CTL to EIAV-infected cells. By using noninfected and EIAV-infected autologous equine kidney (EK) cells in 51Cr-release assays, EIAV-specific cytotoxic activity was detected in unstimulated PBMC from three infected horses. The EIAV-specific cytotoxic activity was major histocompatibility complex (MHC) restricted, as determined by assaying EIAV-infected heterologous EK targets, and was mediated by CD8+ T lymphocytes, as determined by depleting these cells by a panning procedure with an anti-CD8 monoclonal antibody. MHC-restricted CD8+ CTL in unstimulated PBMC from infected horses caused significant specific lysis of autologous EK cells infected with recombinant vaccinia viruses expressing EIAV genes, either env or gag plus 5' pol. The EIAV-specific MHC-restricted CD8+ CTL were detected in two EIAV-infected horses within a few days after plasma viremia occurred and were present after viremia was terminated. The detection of these immune effector cells in EIAV-infected horses permits further studies to determine their in vivo role.     

Gag Protein Epitopes Recognized by ELA-A-Restricted Cytotoxic T Lymphocytes from Horses with Long-Term Equine Infectious Anemia Virus Infection

Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.Equine infectious anemia virus (EIAV) belongs to the Lentivirus genus, which includes human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and several other animal viruses. EIAV causes disease in horses which is characterized by recurrent febrile episodes associated with viremia, anemia, and thrombocytopenia (10). Most infected horses are able to eventually control the disease and become lifelong EIAV carriers (9). The ability of horses to restrict EIAV replication to very low levels and to remain free of clinical disease provides an opportunity to determine the immunologic mechanisms involved in this lentivirus control.Immune responses are required for the termination of the acute viremia during EIAV infection since foals with severe combined immunodeficiency cannot control the initial viremia following EIAV infection, in contrast to normal foals (41). Results suggesting that immune responses are involved in the control of EIAV in carrier horses include the observation that corticosteroid- and cyclophosphamide-treated carrier horses have recurrent viremia and disease (24). Neutralizing antibody can be an important component of the protective immune response against lentiviral infections (12). Type-specific neutralizing antibody appears following the episodes of plasma viremia in EIAV-infected horses (25); however, there is evidence suggesting that the presence of the neutralizing antibody does not necessarily relate to the occurrence and control of viremic episodes (8, 25). Detectable neutralizing antibodies to the variant isolated during a disease episode can appear after the episode is controlled (8). Neutralizing antibody-escape variants are isolated from EIAV carrier horses as early as 5 days after corticosteroid treatment, when the antibody levels have not significantly changed (24). Further, the viremic episode induced by corticosteroid treatment can be terminated before the appearance of neutralizing antibody to the variant causing viremia (24). Other evidence implicating immune responses other than neutralizing antibody in EIAV control includes the following: (i) EIAV carrier horses can resist challenge with a heterologous strain in the absence of detectable neutralizing antibody to the challenge virus (23), and (ii) some horses immunized with an inactivated virus vaccine resist homologous strain challenge without detectable levels of neutralizing antibody but with virus-specific cell-mediated immune responses (17).Accumulating evidence suggests that major histocompatibility complex (MHC) class I-restricted virus-specific cytotoxic T lymphocytes (CTL) may play an important role in the immune control of diseases caused by HIV-1 and SIV infection (5, 26, 51). CTL appear to be involved in both the clearance of the primary viremia in HIV-1 infection (26) and the prevention of disease progression to AIDS (42). In EIAV infection, the appearance of activated CD8⁺ CTL (effectors) correlated with the control of the initial viremic episodes (33). Although the CTL effectors decline to low levels when plasma viremias become undetectable, a high frequency of memory CTL (CTLm) has been detected in some carrier horses (34), and these CTLm recognize either EIAV Env or Gag/Pr proteins or both (15, 34). Both CD8⁺ and CD4⁺ CTL activities have been detected in some EIAV-infected horses (15), but their roles in disease control are not known.The epitopes recognized by CD8⁺ CTL are usually peptides of 8 to 11 amino acids (aa) presented by MHC class I molecules on the target cell surface. Identifying the CTL epitopes and the MHC class I molecules that restrict responses is necessary in order to determine how CTL are involved in the control of disease and to stimulate CTL by vaccination. However, the occurrence of escape mutants which are no longer recognized by CTL is one of the major difficulties for inducing effective CTL responses against different variants (6). Gag protein epitopes recognized by CTL may be of importance because Gag proteins are relatively conserved among EIAV strains (21, 32, 40, 48). In this study, at least 12 peptides with CTL epitopes were recognized by stimulated peripheral blood mononuclear cells (PBMC) from six long-term EIAV-infected horses with different ELA-A alleles. These peptides were identified by using retroviral vectors expressing individual Gag proteins and synthetic overlapping peptides from recognized proteins. We identified two nonamer peptides, one apparently restricted by ELA-A5.1, and another by ELA-A9, molecules.     

Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.

We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl termini of the glycoprotein molecule. Analysis of peptide reactivities with longitudinal serum samples taken from experimentally infected ponies revealed that antibody responses to conserved B-cell determinants appeared earlier and at higher titers than do antibodies specific for determinants contained in the variable domain of gp90. These observations suggest an evolution of antibody responses in EIAV-infected ponies that may correspond to the establishment of immunological control of virus replication and disease routinely observed in EIAV infections. In addition, the mapping of monoclonal antibody epitopes to peptides of 9 to 12 amino acids demonstrated that all of the neutralizing epitopes are located in the variable domain of gp90. The arrangement of neutralizing epitopes and critical structural considerations suggest that EIAV gp90 contains a principal neutralizing domain similar to the V3 loop of human immunodeficiency virus type 1. These antigenic analyses provide an important foundation for further analyzing the protective immune response generated during persistent EIAV infections and also provide potential peptide substrates for diagnostic assays and for vaccine strategies.     

Presentation and binding affinity of equine infectious anemia virus CTL envelope and matrix protein epitopes by an expressed equine classical MHC class I molecule

Control of a naturally occurring lentivirus, equine infectious anemia virus (EIAV), occurs in most infected horses and involves MHC class I-restricted, virus-specific CTL. Two minimal 12-aa epitopes, Env-RW12 and Gag-GW12, were evaluated for presentation by target cells from horses with an equine lymphocyte Ag-A1 (ELA-A1) haplotype. Fifteen of 15 presented Env-RW12 to CTL, whereas 11 of 15 presented Gag-GW12. To determine whether these epitopes were presented by different molecules, MHC class I genes were identified in cDNA clones from Arabian horse A2152, which presented both epitopes. This horse was selected because it is heterozygous for the SCID trait and is used to breed heterozygous females. Offspring with SCID are used as recipients for CTL adoptive transfer, and normal offspring are used for CTL induction. Four classical and three putative nonclassical full-length MHC class I genes were found. Human 721.221 cells transduced with retroviral vectors expressing each gene had equine MHC class I on their surface. Following peptide pulsing, only cells expressing classical MHC class I molecule 7-6 presented Env-RW12 and Gag-GW12 to CTL. Unlabeled peptide inhibition of (125)I-labeled Env-RW12 binding to 7-6-transduced cells demonstrated that Env-RW12 affinity was 15-fold higher than Gag-GW12 affinity. Inhibition with truncated Env-RW12 demonstrated that amino acid positions 1 and 12 were necessary for binding, and single substitutions identified positions 2 and 3 as possible primary anchor residues. Since MHC class I 7-6 presented both epitopes, outbred horses with this allele can be immunized with these epitopes to optimize CTL responses and evaluate their effectiveness against lentiviral challenge.     

Detection and induction of equine infectious anemia virus-specific cytotoxic T-lymphocyte responses by use of recombinant retroviral vectors

Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.     

The simian immunodeficiency virus envelope glycoprotein contains two epitopes presented by the Mamu-A*01 class I molecule.

Cytotoxic T lymphocyte (CTL) responses against the simian immunodeficiency virus (SIV) envelope and Gag proteins were monitored in a Mamu-A*01-positive rhesus macaque infected with SIVsmE660. Peripheral blood mononuclear cells (PBMC) cultured with synthetic peptides spanning the entire gp160 and Gag coding region recognized a total of three epitopes. One located in Gag was identified as the previously described Mamu-A*01-restricted p11cC-->M epitope (CTPYDINQM). The other two epitopes, designated p15m and p54m, were located in the gp160 envelope protein. Both were nine amino acids in length and were predicted to bind Mamu-A*01 because they contained proline and leucine residues at positions 3 and 9, respectively. Indeed, expression of this class I major histocompatibility complex molecule was required for target cell recognition by envelope-specific CD8(+) T cells directed against both epitopes. These Mamu-A*01-restricted epitopes in the SIV envelope will be useful for monitoring immune responses in vaccinated or infected animals.     

Adaptive immunity is the primary force driving selection of equine infectious anemia virus envelope SU variants during acute infection

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infection in horses. The appearance of antigenically distinct viral variants during recurrent viremic episodes is thought to be due to adaptive immune selection pressure. To test this hypothesis, we evaluated envelope SU cloned sequences from five severe combined immunodeficient (SCID) foals infected with EIAV. Within the SU hypervariable V3 region, 8.5% of the clones had amino acid changes, and 6.4% had amino acid changes within the known cytotoxic T lymphocyte (CTL) epitope Env-RW12. Of all the SU clones, only 3.1% had amino acid changes affecting potential N-linked glycosylation sites. In contrast, a much higher degree of variation was evident in SU sequences obtained from four EIAV-infected immunocompetent foals. Within V3, 68.8% of the clones contained amino acid changes, and 50% of the clones had amino acid changes within the Env-RW12 CTL epitope. Notably, 31.9% of the clones had amino acid changes affecting one or more glycosylation sites. Marked amino acid variation occurred in cloned SU sequences from an immune-reconstituted EIAV-infected SCID foal. Of these clones, 100% had amino acid changes within V3, 100% had amino acid changes within Env-RW12, and 97.5% had amino acid changes affecting glycosylation sites. Analysis of synonymous and nonsynonymous nucleotide substitutions revealed statistically significant differences between SCID and immunocompetent foals and between SCID foals and the reconstituted SCID foal. Interestingly, amino acid selection at one site occurred independently of adaptive immune status. Not only do these data indicate that adaptive immunity primarily drives the selection of EIAV SU variants, but also they demonstrate that other selective forces exist during acute infection.     

Selecting peptides to optimize Th1 responses to an equine lentivirus using HLA-DR binding motifs and defined HIV-1 Th peptides

Three moderately to broadly recognized equine infectious anemia virus (EIAV) peptides that contained helper T-lymphocyte (Th) 1 epitopes were previously identified. Although lipopeptide immunization was only weakly immunostimulatory in a preliminary study, as measured by T-lymphocyte proliferation responses, it was of interest to define additional broadly recognized Th1 epitopes to include in future immunization trials. Using broadly cross-reactive and conserved Th epitopes known in the related human immunodeficiency virus-1 (HIV-1) and binding motifs defined in human leukocyte antigen DR molecules as guides, this work identified three new peptides containing Th1 epitopes recognized by 60–75% of EIAV infected horses. The observed similarity across species of major histocompatibility complex (MHC) class II binding motifs and the conservation of Th peptides between related viruses should allow easier targeting of Th epitope regions in less well characterized pathogens and/or in species whose MHC class II molecules are poorly defined.     

T细胞IFN-g表达水平检测方法的建立及其在马传染性 贫血疫苗免疫机理研究中的应用

[目的]马传染性贫血病毒(EIAV)弱毒疫苗致弱机制和免疫保护机理的研究可以为慢病毒疫苗的研究提供重要的模型.为探讨IFN-γ表达水平与疫苗保护性免疫的关系,本研究旨在建立一种准确、有效地检测EIAV感染马不同T细胞亚型表达IFN-γ水平的方法.[方法]我们将分离的马传贫弱毒疫苗免疫马(FDDV)、强毒感染马(LV)和健康马的外周血单核细胞(PBMC),体外分别经病毒(FDDV)和PMA/Inomycin激活、 BFA 阻断蛋白分泌、荧光标记马的特异性表面抗体和IFN-γ抗体等过程后,进行流式检测.[结果]疫苗免疫马产生的特异性IFN-γ水平为CD4 1.7(0.9%/CD8 6.1(1.2%,而强毒组则为CD4 0.6(0.1%/CD8 2.4(0.9%.[结论]本研究建立的多荧光参数流式细胞术同时检测细胞内IFN-γ染色和淋巴细胞亚型的方法,具有良好的特异性,稳定性和重复性.为研究EIAV弱毒疫苗免疫保护机制奠定了基础.     

Antigenic analysis of equine infectious anemia virus (EIAV) variants by using monoclonal antibodies: epitopes of glycoprotein gp90 of EIAV stimulate neutralizing antibodies.

Monoclonal antibodies produced against the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV), a lentivirus, were studied for reactivity with the homologous prototype and 16 heterologous isolates. Eighteen hybridomas producing monoclonal antibodies (MAbs) were isolated. Western blot (immunoblot) analyses indicated that 10 were specific for the major envelope glycoprotein (gp90) and 8 for the transmembrane glycoprotein (gp45). Four MAbs specific to epitopes of gp90 neutralized prototype EIAV infectivity. These neutralizing MAbs apparently reacted with variable regions of the envelope gp90, as evidenced by their unique reactivity with the panel of isolates, suggesting recognition of at least three different neutralization epitopes. The conformation of these epitopes appears to be continuous, as they resisted treatment with sodium dodecyl sulfate and reducing reagents. Monoclonal antibodies that reacted with conserved epitopes on gp90 or gp45 failed to neutralize EIAV. Our data also demonstrated that there was a large spectrum of possible EIAV serotypes and confirmed that antigenic variation occurs with high frequency in EIAV. Moreover, the data showed that variation is a rapid and random process, as no pattern of variant evolution was evident by comparison of 13 isolates from parallel infections. These results represent the first production of neutralizing MAbs specific for a lentivirus glycoprotein and document alterations in one or more neutralization epitopes of the major surface glycoprotein among sequential isolates of EIAV recovered during persistent infection.     

Epitope mapping of monoclonal antibodies against human immunodeficiency virus type 1 structural proteins by using peptides.

Murine monoclonal antibodies directed against the structural proteins p17 and p24 of human immunodeficiency virus type 1 were investigated in an epitope mapping system. Overlapping peptides consisting of 15 amino acids of the p17 and p24 protein, respectively, were used as competitors in an enzyme-linked immunosorbent assay. Three different immunogenic regions (A, B, and C) could be defined, one on p17 and two on p24. Twenty monoclonal antibodies reacted with the human immunodeficiency virus type 1 peptides of region B, although differences in the reactivity of these antibodies with human immunodeficiency virus type 2 and simian immunodeficiency virus strain mac were detectable. Recognized epitopes were characterized by computer analysis as described by T.P. Hopp and K.R. Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981) and P.Y. Chou and G.D. Fasman (Biochemistry 13:222-245, 1974).     

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.     

CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing

It is becoming increasingly clear that any human immunodeficiency virus (HIV) vaccine should induce a strong CD8(+) response. Additional desirable elements are multispecificity and a focus on conserved epitopes. The use of multiple conserved epitopes arranged in an artificial gene (or EpiGene) is a potential means to achieve these goals. To test this concept in a relevant disease model we sought to identify multiple simian immunodeficiency virus (SIV)-derived CD8(+) epitopes bound by a single nonhuman primate major histocompatibility complex (MHC) class I molecule. We had previously identified the peptide binding motif of Mamu-A*01(2), a common rhesus macaque MHC class I molecule that presents the immunodominant SIV gag-derived cytotoxic T lymphocyte (CTL) epitope Gag_CM9 (CTPYDINQM). Herein, we scanned SIV proteins for the presence of Mamu-A*01 motifs. The binding capacity of 221 motif-positive peptides was determined using purified Mamu-A*01 molecules. Thirty-seven peptides bound with apparent K(d) values of 500 nM or lower, with 21 peptides binding better than the Gag_CM9 peptide. Peripheral blood mononuclear cells from SIV-infected Mamu-A*01(+) macaques recognized 14 of these peptides in ELISPOT, CTL, or tetramer analyses. This study reveals an unprecedented complexity and diversity of anti-SIV CTL responses. Furthermore, it represents an important step toward the design of a multiepitope vaccine for SIV and HIV.     

Type 1 CD4(+) T-cell help is required for induction of antipeptide multispecific cytotoxic T lymphocytes by a lipopeptidic vaccine in rhesus macaques

We have optimized the induction of antiviral cytotoxic T lymphocytes (CTL) in rhesus macaques by a lipopeptide vaccine containing seven peptides from simian immunodeficiency virus (SIV) Nef and Gag proteins and a strong T-helper peptide from tetanus toxoid (TT) that is promiscuous in humans (peptide TT 830-846). Two of the eight immunized macaques showed T-helper (Th) cell proliferation and a specific synthesis of gamma interferon in response to TT 830-846 peptide. They also showed multispecific cytotoxic activity against three to five of the immunizing SIV peptides. These results show the importance of a strong specific type 1 Th response for inducing a multispecific CTL response in vivo, which is essential for the development of an anti-human immunodeficiency virus vaccine.     

Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.     

马传染性贫血病毒囊膜基因的研究进展

马传染性贫血病毒是反转录病毒科慢病毒属的成员之一 ,不仅与人免疫缺陷病毒具有序列同源性 ,而且与其血清具有交叉反应。马传染性贫血驴白细胞弱毒疫苗是迄今为止唯一研究成功的慢病毒疫苗。在马传贫病毒囊膜基因的研究中有助于弄清其抗原变异、持续感染和疫苗免疫机理 ,为艾滋病疫苗的研究提供借鉴。对囊膜基因的结构、变异及其在机体免疫应答中的作用进行了讨论。     

Comprehensive screening for human immunodeficiency virus type 1 subtype-specific CD8 cytotoxic T lymphocytes and definition of degenerate epitopes restricted by HLA-A0207 and -C(W)0304 alleles

For this report, the rapid identification and characterization of human immunodeficiency virus type 1 (HIV-1)-derived broadly cross-subtype-reactive CD8 cytotoxic T lymphocyte (CTL) epitopes were performed. Using a gamma interferon (IFN-gamma) Elispot assay-based approach and a panel of recombinant vaccinia viruses expressing gag, env, pol, and nef genes representing the seven most predominant subtypes and one circulating recombinant form of HIV-1, the subtype specificity and cross-subtype reactivity of a CD8 response were directly measured from circulating peripheral blood mononuclear cells (PBMC). Enhanced sensitivity of detection of CD8 responses from cryopreserved PBMC was achieved using autologous vaccinia virus-infected B-lymphoblastoid cell lines as supplemental antigen-presenting cells. Of eleven subjects studied, six exhibited broadly cross-subtype-reactive CD8-mediated IFN-gamma production (at least seven of eight subtypes recognized) to at least one major gene product from HIV-1. Screening of subjects showing broadly cross-subtype-specific responses in the vaccinia virus-based enzyme-linked immunospot (Elispot) assay using a panel of overlapping peptides resulted in the identification of cross-subtype responses down to the 20-mer peptide level in less than 3 days. Three subjects showed broad cross-subtype reactivity in both the IFN-gamma Elispot assay and the standard chromium release cytotoxicity assay. Fine mapping and HLA restriction analysis of the response from three subjects demonstrated that this technique can be used to define epitopes restricted by HLA-A, -B, and -C alleles. In addition, the ability of all three epitopes to be processed from multiple subtypes of their parent proteins and presented in the context of HLA class I molecules following de novo synthesis is shown. While all three minimal epitopes mapped here had previously been defined as HIV-1 epitopes, two are shown to have novel HLA restriction alleles and therefore exhibit degenerate HLA binding capacity. These findings provide biological validation of HLA supertypes in HIV-1 CTL recognition and support earlier studies of cross-subtype CTL responses during HIV-1 infection.     

Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-gamma than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-gamma production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-gamma production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.