P24 Peptides Research Articles

Strategy for Identifying Dendritic Cell-Processed CD4+ T Cell Epitopes from the HIV Gag p24 Protein

Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4+ T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.

Bone induction through controlled release of novel BMP-2-related peptide from PTMC11-F127-PTMC11 hydrogels

Bone morphogenetic protein 2 (BMP-2) is the most powerful osteogenic factor; its effectiveness in enhancing osteoblastic activation has been confirmed both in vitro and in vivo. We developed a novel peptide (designated P24) derived from the ‘knuckle’ epitope of BMP-2 and found it also had osteogenic bioactivity to some extent. The main objective of this study was to develop a controlled release system based on poly(trimethylene carbonate)–F127–poly(trimethylene carbonate) (PTMC11-F127-PTMC11) hydrogels for the P24 peptide, to promote bone formation. By varying the copolymer concentrations, we demonstrated that P24/PTMC11-F127-PTMC11 hydrogels were an efficient system for the sustained release of P24 over 21–35 days. The P24-loaded hydrogels elevated alkaline phosphatase activity and promoted the expression of osteocalcin mRNA in bone marrow stromal cells (BMSCs) in vitro. Radiographic and histological examination showed that P24-loaded hydrogels could induce more effective ectopic bone formation in vivo than P24-free hydrogels. These results indicate that the PTMC11-F127-PTMC11 hydrogel is a suitable carrier for the controlled release of P24, and is a promising injectable biomaterial for the induction of bone regeneration.

Characterization of Immune Responses to Capsid Protein p24 of Human Immunodeficiency Virus Type 1 and Implications for Detection

To further refine our current nanoparticle-based HIV-1 p24 antigen assay, we investigated immune responses to p24 to identify diagnostically significant immune dominant epitopes (IDEs) in HIV-infected human sera, to address cross-reactivity of anti-p24 antibodies to different subtypes, and to identify new biomarkers that distinguish acute from chronic HIV infection for more accurate incidence estimation. We identified two major linear epitope regions, located in the CypA binding loop and adjacent helices and at the end of the C-terminal domain. Most sera (86%) from acutely HIV-1-infected individuals reacted with multiple peptides, while 60% and 30% of AIDS patient samples reacted with multiple and single peptides, respectively. In contrast, 46% and 43% of chronically HIV-1-infected individuals reacted with one and none of the peptides, respectively, and only 11% reacted with multiple p24 peptides, indicating a progression of immune responses from polyclone-like during acute infection to monoclone-like or a nonresponse to linear epitopes during chronic infection. Anti-p24 antibodies (subtype B) show broad cross-reactivity to different HIV-1 subtypes, and the synergistic action of different combinations of anti-HIV antibodies improves capture and detection of divergent HIV-1 subtypes. Our results indicate that the modified peptide immunoassay is sensitive and specific for the rapid identification of HIV-1 p24 IDEs and for investigation of immune responses to p24 during natural HIV-1 infection. The data provide the foundation for development and refinement of new assays for improved p24 antigen testing as future tools for rapid and accurate diagnosis as part of early intervention strategies and estimations of incidence.

SiRNA Knockdown of PD-L1 and PD-L2 in Monocyte-Derived Dendritic Cells only Modestly Improves Proliferative Responses to Gag by CD8+ T Cells from HIV-1-Infected Individuals

Due to their capacity to elicit and regulate immunity, dendritic cells (DCs) are important targets to improve vaccination. Knowing that programmed death-1 (PD-1) high virus-specific T cells become functionally exhausted during chronic exposure to human immunodeficiency virus-1 (HIV-1), the development of a therapeutic DC-based HIV-1 vaccine might include strategies that downregulate PD-L1 and PD-L2 counter-receptors. After showing that monocyte-derived DCs rapidly upregulated PD-L1 and PD-L2 expression upon maturation with a variety of stimuli, e.g., Toll-like receptor ligands and cytokines, we determined that PD-L1 and PD-L2 expression could be knocked down by electroporation of a single small interfering RNA (siRNA) sequence twice at the monocyte and immature stages of DC development. This knockdown approached completion and was specific and lasting for several days. We then added the PD-L1 and PD-L2 silenced monocyte-derived DCs to peripheral blood mononuclear cells from HIV-1-infected individuals along with pools of 15-mer HIV-1 Gag p24 peptides. However, in cultures from six patients, there was only a modest enhancing effect of PD-L1 and PD-L2 silencing on CD8(+) T cell proliferative responses to the DCs. These findings suggest that, in monocyte-derived DCs, additional strategies than PD-L1 or PD-L2 blockade will be needed to improve the function of PD-1 high T cells.

Preparation and ectopic osteogenesis in vivo of scaffold based on mineralized recombinant human-like collagen loaded with synthetic BMP-2-derived peptide

The ideal bone graft material must be biocompatible, biodegradable, osteoconductive and osteoinductive. In this study, a new biomimetic scaffold based on mineralized recombinant collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA), was prepared and the synthetic P24 peptide derived from BMP-2 was introduced into the porous nHA/RHLC/PLA scaffold to improve its osteoinductive property. The nHA/RHLC/PLA implants loaded with 3 mg, 2 mg, 1 mg and 0 mg P24 peptide were implanted subcutaneously into rats. At the 4th, 8th and 12th weeks after implantation, the rats were sacrificed in batch and the samples were harvested. Their osteogenic capability was detected by CT scan and histological observation. The results indicated that the osteogenic capability of 3 mg, 2 mg and 1 mg of the P24 peptide was superior to the implants without the P24 peptide. There was no significant difference between implants with 3 mg and 2 mg P24 peptide, but the osteogenic capability of the two dosage groups was significantly better than that of the 1 mg group. It was concluded that BMP-2-derived peptide can increase the osteoinduction of nHA/RHLC/PLA scaffold and the P24 peptide induced new bone formation in a dose-dependent manner. The nHA/RHLC/PLA scaffold loaded with the synthetic BMP-2-derived peptide is a kind of ideal scaffold material for bone tissue engineering.

Enhancement of Human Antigen-Specific Memory T-Cell Responses by Interleukin-7 May Improve Accuracy in Diagnosing Tuberculosis

Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-gamma) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-gamma. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-gamma message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-gamma responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-gamma responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R(2) = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-gamma by 39% compared to the level with antigen alone. Increased production of IFN-gamma induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-gamma-based assays might improve TB diagnosis in those populations at highest risk for TB.

HLA Alleles Associated with Slow Progression to AIDS Truly Prefer to Present HIV-1 p24

BackgroundThe mechanism behind the association between human leukocyte antigen (HLA) molecules and the rate of HIV-1 disease progression is still poorly understood. Recent data suggest that “protective” HLA molecules, i.e. those associated with a low HIV-1 viral load and relatively slow disease progression, tend to present epitopes from the Gag capsid protein. Although this suggests that preferential targeting of Gag delays disease progression, the apparent preference for Gag could also be a side-effect of the relatively high immunogenicity of the protein.Methods and FindingsTo separate cause and effect, we predicted HIV-1 epitopes from the whole genome of HIV-1, and found that protective HLA alleles have a true preference for the p24 Gag protein, while non-protective HLA alleles preferentially target HIV-1 Nef. In line with this, we found a significant negative correlation between the predicted affinity of the best-binding p24 epitopes and the relative hazard of HIV-1 disease progression for a large number of HLA molecules. When the epitopes targeted by protective HLA alleles were mapped to the known p24 structure, we found that mutations in these epitopes are likely to disturb the p24 dimer structure, which is expected to severely reduce the fitness of the virus.ConclusionsOur results suggest that the intrinsic preference of different HLA molecules to present p24 peptides explains why some HLA molecules are more protective than others.

A Syntheticgagp24 Epitope Chemically Coupled to BSA Through a Decaalanine Peptide Enhances HIV Type 1 Serodiagnostic Ability by Several Folds

p24 is an immunodominant gag core protein of HIV-1. The synthetic immunodominant epitope of p24 and the recombinant p24 show poor immunoreactivity and specificity, respectively. Their application is, therefore, severely limited in the serodiagnosis of HIV-1, although it is an important marker for early diagnosis. These limitations have been overcome by conjugating the synthetic p24 to BSA through a decaalanine peptide spacer. The engineered p24 shows about 5-fold more efficient immunoreactivity than the synthetic p24, and, at the same time, shows a several fold reduction in nonspecific cross-reactivity as compared to recombinant p24. Our strategy to conjugate the p24 peptide epitope to BSA worked well as a consistent and reliable immunodiagnostic marker. This strategy may also prove useful for the diagnosis of other diseases.

CD4 + T cell targeting of human immunodeficiency virus type 1 (HIV-1) peptide sequences present in vivo during chronic, progressive HIV-1 disease

We previously detected HIV-1 Gag-specific CD4 + T cells recognizing reference strain viral epitopes in subjects with progressive, chronic infection. To test whether these CD4 + T cells persist in vivo by failing to recognize autologous HIV-1 epitopes, we compared autologous plasma HIV-1 p24 nucleotide sequences with targeted HXB.2 strain Gag p24 CD4 + T cell epitopes in nine chronically infected, untreated subjects. In five responding subjects, 10 of 26 HXB.2 strain p24 peptides targeted by CD4 + T cells exactly matched autologous plasma viral sequences. Four subjects with plasma viral loads > 100,000 copies/mL had no measurable p24-specific CD4 + T cell responses despite carrying HIV-1 strains that matched HXB.2 sequences at predicted epitopes. These results show that HIV-1-specific CD4 + T cells can persist in chronic HIV-1 infection despite recognition of epitopes present in vivo. However, with high-level in vivo HIV-1 replication, CD4 + T cells targeting autologous HIV-1 may be non-responsive or absent.

Characterization of CD8+ T‐cell responses in HIV‐1‐exposed seronegative commercial sex workers from Nairobi, Kenya

CD8+ T-lymphocyte responses are crucial to the control of HIV-1; therefore, studying the CD8+ immune response in a naturally resistant population could provide valuable insights into an effective anti-HIV response in healthy uninfected individuals. Approximately 5-10% of the women in the Pumwani Commercial Sex Worker cohort in Nairobi, Kenya, have been highly exposed to HIV-1 yet remain HIV-IgG-seronegative and HIV-PCR negative (HIV(ES)). As IFN-gamma production correlates to cytotoxic function, the CD8+ T-lymphocyte IFN-gamma response to HIV p24 peptides was compared in HIV(ES) and HIV-infected (HIV+) individuals. Almost 40% of the HIV(ES) had a CD8+ IFN-gamma+ response that was five times lower in magnitude than that of the HIV+ group. The breadth of the response in HIV(ES) was very narrow and focused primarily on one peptide that is similar to the protective KK10 peptide. In the HIV+ group, low peripheral CD4+ counts negatively influenced the number of CD8+ cells producing IFN-gamma, which may undermine the ability to control HIV. Overall, many of the HIV(ES) women possess a HIV-1 p24-specific CD8+ IFN-gamma response, providing evidence to the specificity needed for an effective HIV vaccine.

Identification of peptides from human pathogens able to cross-activate an HIV-1-gag-specific CD4+ T cell clone

Antigen recognition by T cells is degenerate both at the MHC and the TCR level. In this study, we analyzed the cross-reactivity of a human HIV-1 gag p24-specific CD4(+) T cell clone obtained from an HIV-1-seronegative donor using a positional scanning synthetic combinatorial peptide library (PS-SCL)-based biometrical analysis. A number of decapeptides able to activate the HIV-1 gag-specific clone were identified and shown to correspond to sequences found in other human pathogens. Two of these peptides activated the T cell clone with the same stimulatory potency as the original HIV-1 gag p24 peptide. These findings show that an HIV-1-specific human T helper clone can react efficiently with peptides from other pathogens and suggest that cellular immune responses identified as being specific for one human pathogen (HIV-1) could arise from exposure to other pathogens.

Expansion and Contraction of HIV-Specific CD4 T Cells with Short Bursts of Viremia, but Physical Loss of the Majority of These Cells with Sustained Viral Replication

Chronic infection with the HIV results in poor HIV-specific CD4 T cell proliferation, but more recent analyses using intracellular cytokine staining demonstrated that IFN-gamma-producing, HIV-specific CD4 T cells can be detected for years in HIV-infected subjects. Because it is not known whether the majority of HIV-specific T cells are lost or become dysfunctional, we examined the kinetics of the T cell response over an extended period of time using a panel of 10 HLA-DR tetramers loaded with HIV p24 peptides. Tetramer+ CD4 T cells were present at a relatively high frequency during acute infection, but the size of these populations substantially contracted following suppression of viral replication. Short-term cessation of antiretroviral therapy resulted in a burst of viral replication and concomitant expansion of tetramer+ CD4 T cells, and these populations again contracted following reinitiation of therapy. The kinetics with which these cell populations contracted were characteristic of effector T cells, a conclusion that was supported by their phenotypic (CCR7-CD45RA-) and functional properties (IFN-gamma+). Continued high-level viremia resulted in the physical loss of the majority of tetramer+ CD4 T cells, and the decline of HIV p24-specific CD4 T cells occurred more rapidly and was more substantial than the reduction of total CD4 T cell numbers. We conclude that the population of HIV p24-specific CD4 T cells is initially responsive to changes in the levels of viral Ags, but that the majority of these cells are lost in a setting of chronic viremia.

Measurement of HIV‐1 CRF02_AG–Specific T Cell Responses Indicates the Dominance of a p24gagEpitope in Blood Donors in Abidjan, Côte d’Ivoire

Characterization of human immunodeficiency virus (HIV)-1-specific immune responses against subtypes circulating in areas where the virus is endemic is critical for the design of candidate vaccines. In Cote d'Ivoire, the most prevalent HIV-1 subtype is CRF02_AG. We detected T cell responses to CRF02_AG consensus p24(gag) or protease peptides in 81% of HIV-1- or HIV-1/2-infected blood donors in Abidjan, Cote d'Ivoire. Both the magnitude and the breadth of interferon- gamma enzyme-linked immunospot responses were inversely correlated with plasma viral load. One frequently recognized peptide in p24(gag) was mapped to the optimal epitope (TPQDLNMML). Further studies of this epitope may be important for the development of HIV-1 vaccines for West Africa and West-Central Africa.

Divergent in vitro and in vivo correlates of HIV-specific T-cell responses during onset of HIV viraemia

Cellular immune responses to HIV-1 have been examined mainly in peripheral blood mononuclear cells (PBMC). During onset of HIV replication and antigenaemia after discontinuation of highly active antiretroviral therapy (HAART), PBMC may theoretically contain HIV-specific T cells that are qualitatively and quantitatively different from specific T cells dominating in the tissues. PBMC responses throughout HIV immunotherapy trials may therefore be skewed during recurrent viraemia. To compare cellular HIV-specific in vitro responses in PBMC during onset of HIV viraemia with corresponding in vivo responses, represented by classical delayed-type hypersensitivity tests (DTH). HIV patients (n = 38), pre-immunized with four HIV-1 p24-like consensus peptides (Vacc-4x) during HAART, were subjected to a 14-week treatment interruption with recurrent HIV viraemia. Proliferative T-cell responses to Vacc-4x p24 peptides, HIV p24 protein, and cytomegalovirus (CMV) proteins were measured in PBMC. Corresponding Vacc-4x peptide DTH were expressed as skin infiltrate areas after 48 h. After 14 weeks without HAART, HIV-1 RNA increased to 72,500 copies/ml (median). The Vacc-4x p24 peptide- and HIV-1 p24 protein-induced T-cell proliferation concurrently decreased by 81 and 93% in PBMC during viraemia (medians, P < or = 0.03), whereas proliferative responses to CMV antigens were stable. In contrast, the Vacc-4x DTH areas, rather tended to increase by 36% (P = 0.08) and contained infiltrates dominated by proliferating T cells and macrophages. Divergent in vitro and in vivo HIV-specific cellular immune responses were found during recurrent HIV viraemia. The clinical relevance of both surrogate markers for HIV-related immune responses should be compared in future studies.

CD4+T Cell Responses in HIV‐Exposed Seronegative Women Are Qualitatively Distinct from Those in HIV‐Infected Women

The immune response of human immunodeficiency virus (HIV)-exposed seronegative (ESN) women may be qualitatively different from that in those infected with HIV (HIV(+)). In a cohort of female commercial sex workers in Nairobi, Kenya, we found significantly lower (P< or =.01) levels of CD4(+)-specific immune activation and apoptosis in the ESN women compared with those in the HIV(+) women. Compared with the HIV(+) women, a lower proportion of the ESN women showed p24 peptide pool responses by the short-term, CD4(+)-specific, interferon (IFN)- gamma intracellular cytokine staining assay, whereas the proportion showing responses by the long-term, CD8(+)-depleted T cell proliferation assay was similar. Interestingly, the ESN responders had a 4.5-fold stronger proliferation response (P=.002) than the HIV(+) group. These data suggest that, compared with those in HIV(+) women, CD4(+) T cells in ESN women have a much greater ability to proliferate in response to p24 peptides.

Linker peptide and affinity tag for detection and purification of single-chain Fv fragments

The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs.

Generating neutralizing antibodies, Th1 response and MHC non restricted immunogenicity of HIV-I env and gag peptides in liposomes and ISCOMs with in-built adjuvanticity

For enhancing immunogenicity and develop vaccine strategies using peptide based constructs against HIV-1, a chimeric peptide containing V3 loop and transmembrane sequence of gp41 with two glycine motifs as spacer was constructed. The V3-gp41, gp41 peptide and p17 and p24 peptides separately or in a cocktail were entrapped with or without MA729 as an immunoadjuvant in liposomes or ISCOMs. The immunogenicity, antigen induced T-cell proliferation and cytokine profiles of various formulations were studied in four different inbred strains of mice of H-2d, H-2b, H-2k and H-2q haplotypes, keeping alum as a control adjuvant. Both liposomes and ISCOM preparations elicited high titer and long lasting antibody response (60 days and above). When compared to the alum formulation, the liposomes co-entrapped with MA729 produced high antibody levels, comparable with that induced by ISCOMs. Peptide in alum, liposomes and ISCOMs enhanced both antigen specific IgG2a and IgG2b isotypes and high T-cell stimulation index. Peptide formulations also induced antibodies with high affinity and in vitro neutralizated the formation of HIV-1 syncytia. T-cell supernatants contained high levels of IFN-γ and IL-2. Thus formulation in these adjuvants induced a predominant Th1 like response with MA729 as a versatile novel delivery vehicle for stimulating the appropriate arm of the immune response that can selectively modulate MHC class I or MHC class II response. The above peptide can be of wide vaccination interest as a means to improve immune responses to several other HIV-1 antigens and may serve as candidates for vaccine development.

A balanced type 1/type 2 response is associated with long-term nonprogressive human immunodeficiency virus type 1 infection.

Previous reports have emphasized the requirements for strong type 1 cell-mediated responses in the control of human immunodeficiency virus type 1 (HIV-1). HIV-1 Gag p24-specific CD4 helper T-lymphocyte (HTL) responses have been shown to inversely correlate with viral burden in HIV-1-infected individuals. In this study, peripheral blood mononuclear cells from 70 individuals with chronic progressive HIV-1 infection (clinical progressors), 10 clinical nonprogressors, and 3 immunologically discordant progressors were assessed for HTL proliferation and type 1/type 2 cytokine production. Clinical progressors lacked functional HIV-1-specific HTLs with proliferative and cytokine-producing capacity. Clinical nonprogressors were found to respond to a wide range of HIV-1 antigens from different clades, producing both type 1 and type 2 cytokines. Immunologically discordant progressors responded strongly to clade B Gag p24 with a type 1 cytokine profile but not to other antigens. Thus, in contrast to clinical nonprogressors, neither progressors nor immunologically discordant progressors secreted interleukin-4 (IL-4) in response to HIV-1 antigens. Both clinical nonprogressors and immunologically discordant progressors responded broadly to B clade Gag p24-overlapping peptides. However, IL-4 production in the nonprogressors was restricted to a limited number of p24 peptides. No HIV-1-specific T-cell responses were seen in 20 seronegative controls. Additionally, we observed a rapid type 1 to type 2 shift in the response of one immunologically discordant progressor upon onset of clinical symptoms. These results suggest that a balanced type 1/type 2 profile correlates with successful long-term control of HIV-1.

Magnitude of functional CD8+ T-cell responses to the gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma.

The importance of CD8+ T-cell responses in the control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated, yet few studies have been able to correlate these responses with markers of HIV-1 disease progression. This study measured cell-mediated immune responses using peripheral blood mononuclear cells (PBMC) obtained from 27 patients with chronic HIV-1 infection, the majority of whom were off antiretroviral therapy. The ELISPOT assay was used to detect gamma interferon-secreting PBMC after stimulation with overlapping HIV-1 peptides spanning the Gag, Pol, Env, and Nef proteins in addition to the baculovirus-derived p24 and gp160 proteins. All volunteers had responses to at least one HIV-1-specific peptide. All but one of the subjects (96%) responded to the Gag peptide pool, and 86% responded to the Pol and/or Nef peptide pools. The magnitude and the breadth of T-cell responses directed to either the Gag or p24 peptide pools correlated inversely with viral load in plasma (r = -0.60, P < 0.001 and r = -0.52, P < 0.005, respectively) and directly with absolute CD4+ T-cell counts (r = 0.54, P < 0.01 and r = 0.39, P < 0.05, respectively) using the Spearman rank correlation test. Responses to the Pol and integrase peptide pools also correlated with absolute CD4+ T-cell counts (r = 0.45, P < 0.05 and r = 0.49, P < 0.01, respectively). No correlation with markers of disease progression was seen with specific T-cell responses directed toward the Env or Nef peptides. These data serve as strong evidence that major histocompatibility complex class I presentation of Gag peptides is an essential feature for any HIV-1 vaccine designed to elicit optimal CD8+ T-cell responses.

Synthetic peptide-based electrochemiluminescence immunoassay for anti-Borna disease virus p40 and p24 antibodies in rat and horse serum.

Borna disease virus (BDV) is a neurotropic pathogen that infects a wide variety of vertebrates. We have developed a new electrochemiluminescence immunoassay (ECLIA) for the detection of antibodies to BDV, using three synthetic peptides corresponding to the amino acid residues 3-20 and 338-358 of p40 and 59-79 of p24 peptide of BDV. Using the ECLIA, we examined serum samples for the presence of anti-BDV antibodies in 20 rats (experimentally BDV-infected and uninfected) and 38 horses (13 US horses, experimentally infected and uninfected, and 25 Japanese horses, feral and domestic). The ECLIA, performed in a double-blind manner, detected anti-BDV antibodies in rats with a history of BDV infection, giving results that were in agreement with indirect immunofluorescence assay and/or Western blot (WB) analysis. The ECLIA also correctly identified all three experimentally infected horses and four domestic American horses that were seropositive for BDV antibodies by WB. Among the Japanese horses, at least two out of 10 feral and six out of 15 domestic horses were seropositive for BDV. In most of these cases, the specificity of immunoreactivity was verified by blocking with soluble p40 and p24 peptides.