Recognition of HIV glycoprotein gp120 by T cells. Role of monocyte CD4 in the presentation of gp120

The HIV envelope glycoprotein gp120 binds with high affinity to CD4 and is responsible for the tropism of HIV for CD4+ T cells and monocytes. Efforts to develop HIV vaccines have focused on gp120 and, therefore, a detailed molecular understanding of human immune responses to gp120 is essential. In this report, we have used human T cell clones specific for gp120 to examine the processing and presentation of gp120 to T cells. In particular, we examined the role of the CD4 that is expressed at low levels on the surfaces of human monocytes in the presentation of gp120 by monocytes. The presentation of gp120 to gp120-specific human T cell clones was blocked by pretreatment of monocytes with anti-CD4 mAb. Blocking of monocyte CD4 with anti-CD4 did not inhibit presentation of other Ag or of synthetic peptides representing epitopes within gp120 recognized by gp120-specific T cell clones. These results indicated that the anti-CD4-mediated inhibition occurred at the level of the monocyte, was specific for the gp120 response, and was operative at the initial Ag uptake phase of the Ag-processing pathway. Definitive confirmation that monocyte CD4 functions in the initial uptake step of the gp120-processing pathway was obtained by using soluble CD4 to block the interaction of gp120 with monocyte CD4. These results demonstrate that gp120 expressed by human monocytes plays an important role in the initial uptake of gp120 by monocytes and that gp120 taken up via CD4 is subsequently processed to allow for exposure of epitopes recognized by gp120-specific human T cells. At limiting gp120 concentrations, uptake via CD4 is essential for the presentation of gp120.     

Priming with T helper cell epitope peptides enhances the antibody response to the envelope glycoprotein of HIV-1 in primates.

The induction of a memory immune response to HIV, mediated by any kind of effector mechanism, requires the induction of T cell help. In previous studies performed in different murine MHC haplotypes, three immunodominant T cell epitopes (T1, T2, and TH4.1) had been identified in the HIV envelope glycoprotein. Moreover, these peptides were proliferative T cell epitopes in humans. In this study, rhesus monkeys, Macaca mulatta, were primed with these three peptides either in combination or given separately. Half of the monkeys had a proliferative response to one or more of the priming peptide(s). Those monkeys who had a T cell proliferative response also had a high antibody response after one boost with a suboptimal dose of the native protein gp 160, whereas three of four control monkeys who had received only the native protein immunization gave no detectable antibody response, and one displayed a very weak response. For reasons that are unclear, antibodies only to the gp41 portion of gp 160 could be detected in the sera. Thus, the peptides can prime Th cells in primates for an enhanced antibody response on first exposure to the whole protein. The three peptides belong to highly conserved and nonglycosylated regions of the envelope protein. The fact that the peptides acted as immunogenic T cell proliferative and helper epitopes in nonhuman primates is very encouraging for including them in future vaccine studies in humans.     

Allocation of helper T-cell epitope immunodominance according to three-dimensional structure in the human immunodeficiency virus type I envelope glycoprotein gp120

The specificity and intensity of CD4(+) helper T-cell responses determine the effectiveness of immune effector functions. Promiscuously immunodominant helper T-cell epitopes in the human immunodeficiency virus (HIV) envelope glycoprotein gp120 could be important in the development of broadly protective immunity, but the underlying mechanisms of immunodominance and promiscuity remain poorly defined. In this study, gp120 helper T-cell epitopes were systematically mapped in CBA/J and BALB/c mice by restimulation assays using a set of overlapping peptides spanning the entire sequence of the gp120 encoded by HIV strain 89.6. The results were analyzed in the context of the HIV gp120 structure determined by x-ray crystallography. One major finding was that all of the promiscuously immunodominant gp120 sequences are located in the outer domain. Further analyses indicated that epitope immunogenicity in the outer domain correlates with structural disorder in adjacent N-terminal segments, as indicated by crystallographic B-factors or sequence divergence. In contrast, the correlation was poor when the analysis encompassed the entire gp120 sequence or was restricted to only the inner domain. These findings suggest that local disorder promotes the processing and presentation of adjacent epitopes in the outer domain of gp120 and therefore reveal how three-dimensional structure shapes the profile of helper T-cell epitope immunogenicity.     

Genetic variability in HIV-1 gp120 affects interactions with HLA molecules and T cell receptor

The propensity of HIV-1 to undergo sequence variation, particularly in the envelope glycoprotein gp120, complicates vaccine development and may enable the virus to evade ongoing immune responses in infected individuals. We present here a molecular analysis of the effects of this variability on human T cell recognition of HIV-1 gp120. Synthetic peptides representing a defined CD4+ human T cell epitope in gp120 were used to survey gp120 molecules from various HIV-1 strains for the capacity to be recognized in the context of a single human MHC molecule, DR4. Variation affected recognition at two levels. For some strains, variation in this epitope was sufficient to alter the interaction of Ag receptors on gp120-specific human T cell clones with peptide-DR4 complexes on APC. In the case of two strains, the natural variation was sufficient to prevent the critical initial interaction between the relevant gp120 peptides and DR4 on the APC. However, these strains were highly divergent from the reference strain. Thus it is encouraging to note that the range of natural sequence variation in this T cell epitope falls, for the most part, within the range of peptide sequences that can be accommodated by the relevant human MHC molecule.     

Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes

In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.     

Increased immunogenicity of human immunodeficiency virus gp120 engineered to express Galalpha1-3Galbeta1-4GlcNAc-R epitopes

The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV) envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these carbohydrate chains with Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitopes. These epitopes are ligands for the natural anti-Gal antibody constituting approximately 1% of immunoglobulin G in humans. We hypothesize that vaccination with gp120 expressing alpha-gal epitopes (gp120(alphagal)) results in in vivo formation of immune complexes with anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction between the Fc portion of the antibody and Fcgamma receptors on APC. This in turn results in effective transport of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC for eliciting a strong anti-gp120 immune response. This hypothesis was tested in alpha-1,3-galactosyltransferase knockout mice, which produce anti-Gal. Mice immunized with gp120(alphagal) produced anti-gp120 antibodies in titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120 and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized with gp120(alphagal) than in mice immunized with gp120. It is suggested that gp120(alphagal) can serve as a platform for anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120(alphagal), thereby creating effective prophylactic vaccines.     

N-glycosylation of HIV-gp120 may constrain recognition by T lymphocytes.

The HIV envelope protein gp120 is heavily glycosylated, having 55% of its molecular mass contributed by N-linked carbohydrates. We investigated the role of N-glycosylation in presentation of HIV-gp120 to T cells. T cell clones obtained from humans immunized with a recombinant nonglycosylated form of HIV-gp120 (env 2-3) were studied for their ability to recognize both env 2-3 and glycosylated gp120. We found that 20% of CD4+ T cell clones specific for env 2-3 fail to respond to glycosylated gp120 of the same HIV isolate. Using synthetic peptides, we mapped one of the epitopes recognized by such clones to the sequence 292-300 (NESVAINCT), which contains two asparagines that are glycosylated in the native gp120. These findings suggest that N-linked carbohydrates within an epitope can function as hindering structures that limit Ag recognition by T lymphocytes.     

A Strategy for Eliciting Antibodies against Cryptic,Conserved, Conformationally Dependent Epitopes of HIV Envelope Glycoprotein

Background

Novel strategies are needed for the elicitation of broadly neutralizing antibodies to the HIV envelope glycoprotein, gp120. Experimental evidence suggests that combinations of antibodies that are broadly neutralizing in vitro may protect against challenge with HIV in nonhuman primates, and a small number of these antibodies have been selected by repertoire sampling of B cells and by the fractionation of antiserum from some patients with prolonged disease. Yet no additional strategies for identifying conserved epitopes, eliciting antibodies to these epitopes, and determining whether these epitopes are accessible to antibodies have been successful to date. The defining of additional conserved, accessible epitopes against which one can elicit antibodies will increase the probability that some may be the targets of broadly neutralizing antibodies.

Methodology/Principal Findings

We postulate that additional cryptic epitopes of gp120 are present, against which neutralizing antibodies might be elicited even though these antibodies are not elicited by gp120, and that many of these epitopes may be accessible to antibodies should they be formed. We demonstrate a strategy for eliciting antibodies in mice against selected cryptic, conformationally dependent conserved epitopes of gp120 by immunizing with multiple identical copies of covalently linked peptides (MCPs). This has been achieved with MCPs representing 3 different domains of gp120. We show that some cryptic epitopes on gp120 are accessible to the elicited antibodies, and some epitopes in the CD4 binding region are not accessible. The antibodies bind to gp120 with relatively high affinity, and bind to oligomeric gp120 on the surface of infected cells.

Conclusions/Significance

Immunization with MCPs comprised of selected peptides of HIV gp120 is able to elicit antibodies against conserved, conformationally dependent epitopes of gp120 that are not immunogenic when presented as gp120. Some of these cryptic epitopes are accessible to the elicited antibodies.     

Induction of anti-HIV neutralizing antibodies by synthetic peptides.

Two synthetic peptides containing amino acid sequences analogous to the envelope glycoprotein of human T-lymphotropic virus (HTLV) type III (HTLV-III) and lymphadenopathy associated virus (LAV) were produced and used to immunize rabbits. The subsequent rabbit antisera neutralized HTLV-III infectivity in vitro. The two synthetic peptides corresponded to regions associated with the gp120 or gp41 subunits respectively, of human immunodeficiency virus (HIV). This data indicates that at least two neutralizing epitopes are present on the envelope glycoprotein of HIV and these epitopes are associated with two distinct virus envelope glycoproteins. Antisera generated against these peptides neutralized infectivity of two different isolates of HTLV-III. The data is discussed in terms of possible strategy for developing an effective vaccine against the etiologic agents of acquired immune deficiency syndrome (AIDS).     

Galactose receptors and presentation of HIV envelope glycoprotein to specific human T cells.

Recognition of viral Ag and of the envelope glycoprotein of HIV (gp120) in particular by human Th cells is critical in the immune response to the viral Ag which includes antibody production and generation of cytotoxic cells. Procedures to increase antigenicity of gp120 are highly desirable in a vaccine perspective. Therefore, to induce activation of gp120-specific T cells by a liminal dose of Ag we enhanced uptake of gp120 by exploiting the galactose receptors on APC. Terminal sialic acid residues were removed by neuraminidase treatment from the carbohydrate side chains of the heavily glycosylated gp120. Galactose residues were exposed and hence recognized by galactose receptors on APC. The experiments demonstrated that 1) human monocytes and dendritic cells, but not cells of the B lineage, bear galactose receptor; 2) galactose receptors are indeed involved because enhanced presentation is inhibited by galactose and acetylgalactosamine and competed for by other asialoglycoproteins; 3) galactose receptors mediate internalization of Ag in intracellular compartments that intersect the processing and presenting pathways, resulting in activation of specific T cells; 4) antigenicity of gp120 for specific T cells can be enhanced by the exposure of galactose residues.     

Selective functional depletion of HIV gp120 peptides complexed with MHC from antigen-presenting cells engaged with specific T lymphocytes.

Human T cell lines specific for different peptides of HIV envelope glycoprotein gp120 have been used as probes to identify the availability of functional MHC-peptide complexes on APC. MHC-peptide complexes recognized by T cells specific for peptide 24 (amino acids 225-240) are no longer available on the surface of APC after interaction with irradiated (binding nonproliferating) T cells with the same fine specificity. On the contrary, MHC-peptide complexes recognized by T cells specific for peptide 30 (amino acids 285-300) were functionally available and could stimulate T cells with such a specificity. The reciprocal experiment yielded similar results. The same data were also reproduced with another pair of gp120 peptides. These data demonstrate that upon clustering of peptide-specific T cells with presenting cells presentation of the same peptide to a second cohort of T cells with identical specificity is abolished, suggesting that a selective functional depletion of the MHC-peptide complexes engaged with specific T cells occurs at the surface of the presenting cells. The depletion does not affect other MHC molecules complexed with unrelated peptides.     

Construction and characterization of recombinant VLPs and Semliki-Forest virus live vectors for comparative evaluation in the SHIV monkey model

For testing of recombinant virus-like particles (VLPs) in the SHIV monkey model, SIVmac239 Pr56gag precursor-based pseudovirions were modified by HIV-1 gp160 derived peptides. First, well-characterized epitopes from the HIV-1 envelope glycoprotein were inserted into the Pr56gag precursor by replacing defined regions that were shown to be dispensable for virus particle formation. Expression of these chimeric proteins in a baculovirus expression system resulted in efficient assembly and release of non-infectious, hybrid VLPs. In a second approach the HIV-1IIIB external glycoprotein gp120 was covalently linked to an Epstein-Barr virus derived transmembrane domain. Coexpression of the hybrid envelope derivative with the Pr56gag precursor yielded recombinant SIV derived Pr56gag particles with the HIV-1 gp120 firmly anchored on the VLP surface. Immunization of rhesus monkeys with either naked VLPs or VLPs adsorbed to alum induced substantial serum antibody titers and promoted both T helper cell and cytotoxic T lymphocyte responses. Furthermore, priming macaques with the corresponding set of recombinant Semliki-Forest viruses tended to enhance the immunological outcome. Challenge of the immunized monkeys with chimeric SHIV resulted in a clearly accelerated reduction of the plasma viremia as compared to control animals.     

Structure based sequence analysis & epitope prediction of gp41 HIV1 envelope glycoprotein isolated in Pakistan

ABSTRACT: BACKGROUND: Gp41 is an envelope glycoprotein of human immune deficiency virus (HIV). HIV viral glycoprotein gp41, present in complex with gp120, assists the viral entry into host cell. Over eighty thousands individuals are HIV infected in Pakistan which makes about 0.2% of 38.6 million infected patients worldwide. Hence, HIV gp41 protein sequences isolated in Pakistan were analyzed for the CD4 and CD8 T cells binding epitopes. RESULTS: Immunoinformatics tools were applied for the study of variant region of HIV gp41envelope protein. The protein nature was analyzed using freely accessible computational software. About 90 gp41 sequences of Pakistani origin were aligned and variable and conserved regions were found. Four segments were found to be conserved in gp41 viral protein. A method was developed, involving the secondary structure, surface accessibility, hydrophobicity, antigenicity and molecular docking for the prediction and location of epitopes in the viral glycoprotein. Some highly conserved CD4 and CD8 binding epitopes were also found using multiple parameters. The predicted continuous epitopes mostly fall in the conserved region of 1-12; 14-22 and 25-46 and can be used as effective vaccine candidates. CONCLUSION: The study revealed potential HIV subtype a derived cytotoxic T cell (CTL) epitopes from viral proteome of Pakistani origin. The conserved epitopes are very useful for the diagnosis of the HIV 1 subtype a. This study will also help scientists to promote research for vaccine development against HIV 1 subtype a, isolated in Pakistan.     

Role of APC in the selection of immunodominant T cell epitopes

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.     

Cutting edge: competition for APC by CTLs of different specificities is not functionally important during induction of antiviral responses

The hypothesis that T cell competition for access to APC influences priming of CTL responses is a controversial issue. A recent study using OVA as a model Ag supports this hypothesis and received considerable attention. However, using a comparable approach, we reached a different conclusion. We analyzed whether TCR transgenic T cells specific for lymphocytic choriomeningitis virus gp33-41/D(b) could inhibit the priming of endogenous responses against gp33-41 and against two other lymphocytic choriomeningitis virus glycoprotein-derived CTL epitopes. After priming with different stimuli, gp33-41/D(b)-specific TCR transgenic T cells reduced the endogenous gp33-41/D(b) response in a dose-dependent way, but all other endogenous responses were unaffected. Even when >10(6) TCR transgenic cells were combined with weak priming, no reduction of responses other than of those specific for gp33-41/D(b) was observed. Thus, competition for APC by CTLs of different specificities is not of functional relevance in antiviral immune responses.     

CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses

MHC class I-restricted T cell epitopes lack immunogenicity unless aided by IFA or CFA. In an attempt to circumvent the known inflammatory side effects of IFA and CFA, we analyzed the ability of immunostimulatory CpG-DNA to act as an adjuvant for MHC class I-restricted peptide epitopes. Using the immunodominant CD8 T cell epitopes, SIINFEKL from OVA or KAVYNFATM (gp33) from lymphocytic choriomeningitis virus glycoprotein, we observed that CpG-DNA conveyed immunogenicity to these epitopes leading to primary induction of peptide-specific CTL. Furthermore, vaccination with the lymphocytic choriomeningitis virus gp33 peptide triggered not only CTL but also protective antiviral defense. We also showed that MHC class I-restricted peptides are constitutively presented by immature dendritic cells (DC) within the draining lymph nodes but failed to induce CTL responses. The use of CpG-DNA as an adjuvant, however, initiated peptide presenting immature DC progression to professional licensed APC. Activated DC induced cytolytic CD8 T cells in wild-type mice and also mice deficient of Th cells or CD40 ligand. CpG-DNA thus incites CTL responses toward MHC class I-restricted T cell epitopes in a Th cell-independent manner. Overall, these results provide new insights into CpG-DNA-mediated adjuvanticity and may influence future vaccination strategies for infectious and perhaps tumor diseases.     

A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.     

A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120.

Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines.     

The importance of exogenous antigen in priming the human CD8+ T cell response: lessons from the EBV nuclear antigen EBNA1

Mouse models suggest that the processing of exogenous Ag by dendritic cells can be important for priming the CD8(+) CTL response. To study the situation in humans, we have exploited the CTL response to EBV infection. In this context EBV expresses eight latent proteins, of which EBV-encoded nuclear Ag (EBNA) 3A, 3B, and 3C appear to be immunodominant for CTL responses, whereas another nuclear Ag, EBNA1, which is completely protected from endogenous presentation via the MHC class I pathway, is thought to induce responses rarely, if ever. Here, using EBNA1 peptides and/or EBNA1 protein-loaded dendritic cells as in vitro stimuli, we have identified memory CTL responses to HLA-B*3501, -B7, and -B53-restricted EBNA1 epitopes that can be as strong as those seen in immunodominant epitopes from the "conventionally processed" EBNA3 Ags. Furthermore, we used HLA-peptide tetramers to show that the primary response to one such EBNA1 epitope constituted up to 5% of the CD8(+) T cells in infectious mononucleosis blood, the strongest latent Ag-specific response yet detected in this setting. We conclude that exogenous protein represents a significant source of Ag for priming the human CTL response.     

Shaping T Cell – B Cell Collaboration in the Response to Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 by Peptide Priming

Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming.