High-affinity interactions between peptides and heat shock protein 70 augment CD8+ T lymphocyte immune responses.

Exogenously delivered antigenic peptides complexed to heat shock proteins (HSPs) are able to enter the endogenous Ag-processing pathway and prime CD8+ CTL. It was determined previously that a hybrid peptide containing a MHC class I-binding epitope and HSP70-binding sequence Javelin (J0) in complex with HSP70 could induce cytotoxic T cell responses in vivo that were more robust than those induced by the minimal epitope complexed with HSP70. The present study introduces a novel, higher-affinity HSP70-binding sequence (J1) that significantly enhances binding of various antigenic peptides to HSP70. A competition binding assay revealed a dissociation constant that was 15-fold lower for the H2-K(b) OVA epitope SIINFEKL-J1 compared with SIINFEKL-J0, indicating a substantially higher affinity for HSP70. Further, modifying the orientation of the hybrid epitope and introducing a cleavable linker sequence between the Javelin and the epitope results in even greater immunogenicity, presumably by greater efficiency of epitope processing. The enhanced immunogenicity associated with Javelin J1 and the cleavable linker is consistently observed with multiple mouse and human epitopes. Thus, by creating a series of epitopes with uniform, high-affinity binding to HSP70, successful multiple epitope immunizations are possible, with equal delivery of each antigenic epitope to the immune system via HSP70. These modified epitopes have the potential for creating successful multivalent vaccines for immunotherapy of both infectious disease and cancer.

Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response.

2F5 is one of the few broadly neutralizing monoclonal antibodies against type 1 Human Immunodeficiency Virus (HIV-1). It recognizes the amino acid sequence ELDKWAS in gp41. We have previously identified a number of immunotargeting 2F5-reactive candidate immunogens. Three of them (designated H-BT1-3) have the ELDKWAS sequence constrained at beta-turn sites within the immunoglobulin heavy chain. Two others (L-CT and L-CTx3) have the sequence attached at the C-terminus of the immunoglobulin light chain with minimal conformational constraints. In the present investigation, the H-BTs were found to bind 2F5 with up to 10-fold higher affinities than their unconstrained counterpart. When used as immunogens, immunogen-specific antibodies were induced with or without adjuvant, confirming the immunotargeting potential of these immunogen constructs. While HIV-1 gp160 cross-reactive antibodies were induced, virus neutralization was not detected. Thus, factors other than 2F5 binding affinity may have a critical role to play in the design of a 2F5-based vaccine.

Assessing human alloimmunization as a strategy for inducing HIV type 1 neutralizing anti-HLA responses.

Xenovaccination of rhesus macaques with human HLA Class I and II proteins has been demonstrated to elicit protective immunity against challenge with SIV grown in human cells. To determine if alloimmunization in humans could lead to protective immunity against HIV-1, we prospectively followed a small group of women receiving whole-cell alloimmunization in the form of leukocyte immunotherapy for recurrent spontaneous abortion. Whole-cell vaccine recipients and their respective partners (referred to as donors) provided pre- and postimmune blood samples for analysis. Study participants were HLA typed by sequence-specific PCR and antibodies specific for HLA Class I and II antigens were measured in recipient plasma. To determine if anti-HLA antibody responses detected in recipient plasma samples were capable of neutralizing HIV-1 in vitro, we grew laboratory strain HIV-1(IIIB) and primary isolate HIV-1(301660) in donor-derived CD4(+) T lymphocytes. The ability of purified whole IgG from responding patients to neutralizing infectivity of the respective donor-derived virus was then assayed in vitro. All donor-recipient pairs were determined to be HLA discordant for at least one Class I and one Class II locus. Two of seven female recipients in total made strong anti-HLA antibody responses specific to the HLA haplotype of the male donor in response to the alloimmunization regimen. For one recipient, IgG antibodies specific for donor HLA Class I and II antigens were able to neutralize both HIV-1(IIIB) and a primary isolate HIV-1(301660). In addition polyclonal anti-HLA class II antibodies against a single determinant (DR4) of this donor were also neutralizing. In contrast, the other recipient exhibiting antibodies only against donor HLA Class I antigens did not neutralize HIV-1(IIIB). Using samples from a small number of women undergoing leukocyte immunotherapy, we have demonstrated for the first time that allele-specific anti-HLA antibodies elicited through human alloimmunization are capable of neutralizing HIV-1 in vitro.

Cytotoxic T lymphocyte reactivity to gp100, MelanA/MART-1, and tyrosinase, in HLA-A2-positive vitiligo patients.

Vitiligo is a common depigmentation disorder thought to result from autoimmune destruction of melanocytes. Recent studies suggest a role for cell-mediated immune responses to melanocyte differentiation antigens, including gp100, MelanA/MART-1, and tyrosinase, in vitiligo pathogenesis. This study investigated T cell reactivity to MelanA/MART-1, tyrosinase, and gp100, in HLA-A2-positive patients with vitiligo. Melanocyte-specific T cell responses were measured ex vivo via enzyme-linked immunospot assay following stimulation with MelanA/MART-1, tyrosinase, and modified gp100 epitopes. Antigen-specific T lymphocyte reactivity to gp100 peptides was seen in 15 of 17 (88%) patients, with many demonstrating very high reactivity at levels comparable with those observed with common recall antigens. Reactivity to gp100 was noted to be associated with disease activity. Antigen-specific T lymphocyte reactivity to MelanA/MART-1 and tyrosinase peptides was not observed ex vivo in our patients, and only one patient demonstrated responses to MelanA/MART-1 and tyrosinase peptides following in vitro re-stimulation. Our findings implicate T cell reactivity to gp100 in patients with active disease and support the concept of an immunopathologic mechanism in vitiligo, in which cell-mediated responses to normal melanocyte antigens play a crucial part.

Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis.

There is a strong association between seronegative arthritis and HLA B27, but it is still unresolved whether the contribution of B27 to disease pathogenesis is solely as a restriction element for an arthritogenic peptide, or whether B27 itself serves as an autoantigen. This study uses transgenic rats to address the question as to whether exposure to an arthritogenic pathogen can alter tolerance to B27. Unlike their nontransgenic counterparts, B27-transgenic rats are tolerant of B27 immunization using either B27(+) splenocytes or plasmid DNA and do not develop anti-B27 CTL. However, if splenocytes from such immunized animals are exposed to Chlamydia in vitro, CTL are generated that lyse B27(+) targets. No killing was seen with targets transfected with control B7, B14, B40, or B44. This phenomenon was not observed with immunization by nontransgenic splenocytes, or HLA-A2 DNA alone. Using targets expressing mutated B27, we show that the epitope for autoreactive CTL recognition of B27 involves the Lys(70) amino acid residue in the alpha(1) domain of the MHC class I molecule. The generation of CTL with specificity for B27 under these conditions demonstrates that tolerance to B27 can be subverted by CHLAMYDIA: This indicates a dynamic interrelationship between the pathogen and B27, which may have important implications for B27-related spondyloarthropathies triggered by intracellular bacteria.

Construction of recombinant targeting immunogens incorporating an HIV-1 neutralizing epitope into sites of differing conformational constraint.

2F5 is one of the very few monoclonal antibodies with the capacity to neutralize a wide spectrum of type 1 human immunodeficiency virus (HIV-1) strains and primary isolates. Constructing an immunogen that contains a conformational mimic of the epitope recognized by 2F5 could provide the means to induce a broadly neutralizing anti-HIV-1 antibody response. Thus, in an effort to create a targeted, adjuvant-independent immunogen able to induce a 2F5-like antibody response, the gp41 sequence recognized by 2F5 (ELDKWAS) was genetically incorporated into different regions of an antibody specific for a framework determinant on human leukocyte antigen (HLA)-DR. All constructs were expressed, secreted from Sf9 insect cells, and found to retain the anti-HLA-DR specificity of the parental antibody. Three of the four constructs in which the ELDKWAS sequence was incorporated into a beta-turn (BT)-like conformational site were recognized by the 2F5 antibody. In contrast, none of the five constructs with the same sequence incorporated into surface-exposed regions of helical turn had any detectable 2F5 reactivity. In addition to demonstrating the significant plasticity of several regions in the antibody molecule in terms of accepting foreign sequences without loss of expression or binding specificity, these results also suggest that the native epitope recognized by the 2F5 antibody may be more beta-turn-like than helical in conformation. Importantly, with respect to vaccine development, the 2F5-reactive antibody constructs represent candidate immunogens for the adjuvant-independent induction of an HIV-1, neutralizing 2F5-like antibody response in humans.