The effect of an anti-HLA-B27 immune response on CTL recognition of Chlamydia.

The interplay between triggering bacteria and HLA-B27 in the pathogenesis of the spondyloarthropathies remains one of the most active areas of investigation in the rheumatic diseases. This has proved difficult to study systematically in the clinical setting, and in this study we utilized a rat model to address the influence that B27-related immunity may have on the process of generating anti-Chlamydia immunity. When splenocytes from HLA-B27 DNA-immunized Lewis (LEW) animals received restimulation in vitro with Chlamydia-treated cells from B27-transgenic LEW rats, we observed that in addition to the expected CTL recognition of HLA-B27, there was also anti-Chlamydia CTL killing of Chlamydia-sensitized syngeneic fibroblast targets. This was not seen when responding cells in vitro were naive LEW splenocytes. To confirm the existence of CTLs recognizing both HLA-B27 and Chlamydia, LEW rats were immunized with B27-transgenic LEW cells, instead of the B27 DNA construct. Splenocytes from the immune rats were restimulated in vitro with Chlamydia-treated B27-transgenic LEW cells. In this instance, the CTLs retained the allele-specific recognition of HLA-B27, as well as recognition of Chlamydia-sensitized syngeneic fibroblasts. Thus, if there is prior expansion of an immune response against HLA-B27, then the resulting splenocytes demonstrate a reduced threshold for generating a primary anti-Chlamydia CTL response. These studies implicate a dynamic interrelationship between recognition of HLA-B27 and Chlamydia trachomatis. The results may have implications for deciphering the cellular basis of Chlamydia-induced reactive arthritis.

Sequence and peptide-binding motif for a variant of HLA-A*0214 (A*02142) in an HIV-1-resistant individual from the Nairobi Sex Worker cohort.

As part of the ongoing study of natural HIV-1 resistance in the women of the Nairobi Sex Workers' study, we have examined a resistance-associated HLA class I allele at the molecular level. Typing by polymerase chain reaction using sequence-specific primers determined that this molecule is closely related to HLA-A*0214, one of a family of HLA-A2 supertype alleles which correlate with HIV-1 resistance in this population. Direct nucleotide sequencing shows that this molecule differs from A*0214, having a silent nucleotide substitution. We therefore propose to designate it HLA-A*02142. We have determined the peptide-binding motif of HLA-A*0214/02142 by peptide elution and bulk Edman degradative sequencing. The resulting motif, X-[Q,V]-X-X-X-K-X-X-[V,L], includes lysine as an anchor at position 6. The data complement available information on the peptide-binding characteristics of this molecule, and will be of use in identifying antigenic peptides from HIV-1 and other pathogens.

Simian immunodeficiency virus (SIV) infection of a rhesus macaque induces SIV-specific CD8(+) T cells with a defect in effector function that is reversible on extended interleukin-2 incubation.

A vigorous expansion of antigen-specific CD8(+) T cells lacking apparent effector function was observed in a rhesus macaque acutely infected with the simian immunodeficiency virus (SIV) strain SIVmac239. Antigen-specific CD8(+) T cells were identified using antigenic-peptide class I major histocompatibility complex tetramers. As many as 8.3% of CD8(+) cells recognized the Mamu-A*01-associated SIV epitope Gag(181-189) (CTPYDINQM); however, these cells demonstrated no effector function when presented with peptide-incubated targets, as measured by intracellular cytokine staining for gamma interferon (IFN-gamma), interleukin-2 (IL-2) production, or direct cellular lysis. Similar results were observed with three other SIV peptide antigens. Nonresponsiveness did not correlate with apoptosis of the CD8(+) cells, nor were cells from this macaque impaired in their ability to present peptide antigens. Associated with the nonresponsive state was a lack of IL-2 production and decreased IL-2 receptor expression. Exogenous IL-2 treatment for 1 week in the absence of antigenic stimulation restored antigen-specific responses and the quantitative correlation between tetramer recognition and antigen-responsive IFN-gamma secretion. This case report suggests a regulatory mechanism that may impede the effector function of antigen-specific T cells during acute infection with SIV or human immunodeficiency virus in some cases. This mechanism may participate in the failure of the immune system to limit infection.

The HLA A2/6802 supertype is associated with reduced risk of perinatal human immunodeficiency virus type 1 transmission.

Certain HLAs may, in part, account for differences in human immunodeficiency virus type 1 (HIV-1) susceptibility by presenting conserved immunogenic epitopes for T cell recognition. The HLA supertype A2/6802 is associated with decreased susceptibility to HIV-1 among sex workers. The alleles in this supertype present the same HIV-1 peptide epitopes for T cell recognition in some cases. This study sought to determine whether the HLA A2/6802 supertype influenced HIV-1 transmission in a prospective cohort of HIV-1-infected mothers and children in Kenya. Decreased perinatal HIV-1 infection risk was strongly associated with possession of a functional cluster of related HLA alleles, called the A2/6802 supertype (odds ratio, 0.12; 95% confidence interval, 0.03-0.54; P=.006). This effect was independent of the protective effect of maternal-child HLA discordance. These data provide further evidence that HLA supertypes are associated with differential susceptibility to HIV-1 transmission.

Creating HIV-1 reverse transcriptase cytotoxic T lymphocyte target structures by HLA-A2 heavy chain modifications.

Vigorous HIV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) responses play an important role in the control of HIV-1 replication and the induction of a strong, broadly cross-reactive CTL response remains an important goal of HIV vaccine development. It is known that the display of high levels of class I MHC-viral peptide complexes at the cell surface of target cells is necessary to elicit a strong CTL response. We now report two strategies to enhance the presentation of defined HIV-1 epitope-specific CTL target structures, by incorporating subdominant HIV-1 reverse transcriptase (RT) CTL epitope sequences into the human class I MHC molecule HLA-A2. We show that either incorporation of HIV-1 CTL epitopes into the signal sequence of HLA or tethering of epitopes to the HLA-A2 heavy chain provide simple ways to create effective CTL target structures that can be recognized and lysed by human HLA-A2-restricted RT-specific CD8(+) CTL. Moreover, cells expressing these epitope-containing HLA-A2 constructs stimulated the generation of primary epitope-specific CTL in vitro. These strategies offer new options in the design of plasmid DNA-based vaccines or immunotherapeutics for the induction of CTL responses against subdominant HIV-1 epitopes.

Anti-major histocompatibility complex antibody responses in macaques via intradermal DNA immunizations.

In simian immunodeficiency virus (SIV) models, immunization of macaques with uninfected human cells or human major histocompatibility complex (MHC) proteins can induce xenogeneic immune responses which can protect the animals from subsequent SIV challenges. These studies suggest that the induction of anti-MHC immune responses can be a viable vaccine strategy against human immunodeficiency virus type 1 (HIV-1). We have previously shown in mouse studies that DNA immunization with class I and class II MHC-encoding plasmids can elicit both xenogeneic and allogeneic antibody responses against conformationally intact MHC molecules (Vaccine 17 (1999) 2479-92). Here we take these observations one step closer to human applications and report that intradermal needle immunizations of non-human primates with plasmid DNA encoding human MHC alleles can safely elicit xenogeneic anti-MHC antibody responses. Moreover, injecting macaques with DNA encoding a specific macaque allogeneic MHC induced anti-allogeneic MHC antibodies production. These studies show that DNA immunization with MHC-encoding vectors can indeed be used to induce specific anti-human xenogeneic, as well as anti-macaque allogeneic MHC immunity in non-human primates. This strategy could thus be used to mobilize anti-MHC antibody response which may be useful as part of an anti-HIV-1 vaccination approach.

The use of bone marrow-chimeric mice in determining the MHC restriction of epitope-specific cytotoxic T lymphocytes.

Plasmid DNA immunization has emerged as a promising vaccine strategy against infectious agents, as well as a potential intervention for the treatment of cancer, autoimmunity, and allergy (1). Until recently, however, the cellular events by which injected plasmid DNA elicits potent antibody and cytotoxic T-lymphocyte (CTL) responses were largely unknown. Upon intramuscular (i.m.) injection of naked DNA, predominant expression of transfected DNA occurs in the myofibers (2), but no direct transfection of antigen presenting cells (APC) has been reported. There are essentially three different mechanisms by which CTLs can be primed by the injected DNA (3). The first possibility is that the transfected muscle cells directly activate CTLs by presenting the antigenic peptide on their MHC class I molecules. Alternatively, the priming of CTLs may be mediated by professional APC taking up antigen released from muscle cells. Finally, CTL priming may involve direct transfection of APC occurring, albeit at low level, and that the CTLs are activated by the transfected APC.

Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus.

4-1BB ligand (4-1BBL) is a member of the TNF family expressed on activated APC. 4-1BBL binds to 4-1BB (CD137) on activated CD4 and CD8 T cells and in conjunction with strong signals through the TCR provides a CD28-independent costimulatory signal leading to high level IL-2 production by primary resting T cells. Here we report the immunological characterization of mice lacking 4-1BBL and of mice lacking both 4-1BBL and CD28. 4-1BBL-/- mice mount neutralizing IgM and IgG responses to vesicular stomatitis virus that are indistinguishable from those of wild-type mice. 4-1BBL-/- mice show unimpaired CTL responses to lymphocytic choriomeningitis virus (LCMV) and exhibit normal skin allograft rejection but have a weaker CTL response to influenza virus than wild-type mice. 4-1BBL-/-CD28-/- mice retain the CTL response to LCMV, respond poorly to influenza virus, and exhibit a delay in skin allograft rejection. In agreement with these in vivo results, allogeneic CTL responses of CD28-/- but not CD28+/+ T cells to 4-1BBL-expressing APC are substantially inhibited by soluble 4-1BB receptor as is the in vitro secondary response of CD28+ T cells to influenza virus peptides. TCR-transgenic CD28-/- LCMV glycoprotein-specific T cells are insensitive to the presence of 4-1BBL when a wild-type peptide is used, but the response to a weak agonist peptide is greatly augmented by the presence of 4-1BBL. These results further substantiate the idea that different immune responses vary in their dependence on costimulation and suggest a role for 4-1BBL in augmenting suboptimal CTL responses in vivo.

Xenogeneic and allogeneic anti-MHC immune responses induced by plasmid DNA immunization.

Major histocompatibility complex (MHC) proteins are known to be incorporated into the human immunodeficiency virus (HIV-1) envelope as the virion buds from the host cell surface. Studies using simian immunodeficiency virus (SIV) infection of macaques have demonstrated that immunization with uninfected human cells or purified HLA proteins can provide protection from challenge with live SIV when it is grown in human cells expressing the same MHC alleles. Thus the induction of anti-MHC immune responses represents an important option to consider with respect to vaccine design for SIV and HIV. Here we examine plasmid DNA immunization strategies as an alternative to cellular or protein immunogens for the induction of xenogeneic and allogeneic immune responses in C57BL/6 mice and in an HLA transgenic mouse model system, respectively. We compared the immunogenicity of HLA-A2- and HLA-B27-expressing splenocytes with the corresponding plasmid DNA immunogens. Results from the transgenic mouse experiments indicate that plasmid DNA immunization with both class I and class II MHC-encoding vectors can elicit antibody responses recognizing conformationally intact MHC molecules. Our data also show that immunization with class I MHC-encoding DNA immunogens can elicit cytotoxic T-lymphocyte responses, demonstrating the potential to mobilize both antibody and cell-mediated anti-MHC immune responses in the context of this approach to HIV-1 vaccine design.

Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes.

Many CTL epitopes of clinical importance, particularly those derived from tumor Ags, display relatively poor MHC binding affinity and stability. Because in vivo immunogenicity, and thus the efficacy of peptide-based vaccines, is thought to be determined by MHC/peptide complex stability, there is a need to develop a simple strategy for enhancing the binding of suboptimal epitopes. Toward this goal, the ability to enhance suboptimal peptides through covalent linkage to beta2-microglobulin (beta2m) was explored. Two suboptimal variants of a high-affinity Db-restricted influenza nucleoprotein peptide were covalently linked, via a polypeptide spacer, to the amino terminus of human beta2m and the recombinant fusion proteins expressed in Escherichia coli. When compared with their uncoupled counterparts, the beta2m-linked epitopes display enhanced MHC stabilization and antigenicity. Thus, tethering epitopes to beta2m provides a simple method for augmenting the biological activity of suboptimal peptides and could be useful in the design of peptide-based vaccines or immunotherapeutics.

Epitope-specific cytotoxic T lymphocyte induction by minigene DNA immunization.

Plasmid DNA vaccines encoding full-length antigen often induce both potent antibody and cytotoxic T lymphocyte (CTL) responses. Here, we examine strategies to exclusively elicit epitope-specific CTL responses using DNA constructs expressing a minimal class I MHC-restricted epitope of the nucleoprotein (NP) of influenza virus. The effects of the addition of an ER leader sequence or cytokine combination on minigene-induced CTL responses in vivo were assessed following both delivery by needle injection into skeletal muscle and by gene gun bombardment into skin epidermis. Our data indicate that the leader sequence enhanced the magnitude of the CTL responses, whereas co-injection of the cytokine genes IL-12 and GM-CSF had a minimal effect. An antibody response against NP was not observed in any of the mice receiving the minigene constructs.

Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization.

Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50-100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000-30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.

NK cells can recognize different forms of class I MHC.

NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and beta2m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.

Mother-child class I HLA concordance increases perinatal human immunodeficiency virus type 1 transmission.

Major histocompatibility complex (MHC) gene products are expressed on human immunodeficiency virus (HIV)-infected cells and incorporated into the lipid envelope of HIV virions. Macaques immunized with human MHC gene products are protected from simian immunodeficiency virus challenge when the virus is grown in cells expressing the same MHC alleles. To relate these findings to mother-to-child transmission of HIV-1, investigations of whether sharing HLA between mother and infant influenced the risk of transmission of HIV-1 to the child were carried out. Class I HLA concordance was independently associated with a stepwise increase in the risk of perinatal HIV-1 transmission for each additional concordant allele (odds ratio, 2.63; 95% confidence interval, 1.36-5.07; P = .003). Thus, discordant HLA may provide infants with a means of protection against HIV-1 as a result of allogeneic infant anti-maternal MHC immune responses.

Anti-HLA alloantibody is found in children but does not correlate with a lack of HIV type 1 transmission from infected mothers.

Searching for mechanisms of natural resistance to HIV infection with which to guide HIV vaccine design, we have examined antibody responses to HLA class I antigens in children of HIV-1-infected mothers. Anti-HLA antibodies are known to block HIV infectivity in vitro and can be protective against SIV challenge in macaques immunized with purified class I HLA. It was hypothesized that alloantibody to maternal HLA in children might contribute to the prevention of mother-to-child transmission of HIV-1. In fact, a surprisingly high proportion of the children examined, 22%, were found to have antibody against class I alloantigens. This alloantibody, however, did not correlate with the HIV status of the children and was found in a similar proportion of children of HIV-negative mothers. The HLA specificity of the antibody was not correlated with noninherited maternal HLA alleles and occurred with a higher frequency in older children. This result suggests environmental factors, rather than exposure to maternal cells, are involved in the formation of the alloantibody. The finding that anti-allo-class I HLA antibodies are not associated with a decreased risk of mother-to-child transmission indicates that this humoral immune response is unlikely to be the natural mechanism that accounts for the lack of transmission observed in many births. This result, however, does not preclude the further investigation of cellular alloimmune responses, or the use of alloimmunization as an artificial HIV immunization strategy.

Naturally occurring IgG anti-HLA alloantibody does not correlate with HIV type 1 resistance in Nairobi prostitutes.

In an effort to identify an immunological basis for natural resistance to HIV-1 infection, we have examined serum antibody responses to HLA class I antigens in female prostitutes of the Nairobi Sex Workers Study. Anti-HLA antibodies are known to block HIV infectivity in vitro and can be protective against SIV challenge in macaques immunized with purified class I HLA. Thus, it was postulated that broadly cross-reactive alloantibodies recognizing common HLA alleles in the client population might contribute to the prevention of heterosexual transmission of HIV. In fact, 12% of the women were found to have serum IgG antibodies against class I alloantigens. However, this alloantibody did not correlate with the HIV status of the women and was found in a similar proportion of HIV-positive and HIV-resistant women. The observed levels of alloantibody did not increase with HIV infection in susceptible individuals, suggesting that potential antigenic mimicry between HIV and host HLA class I antigens does not significantly increase levels of anti-class I antibodies. The lack of correlation between serum anti-allo-class I HLA antibodies and the risk of sexual transmission indicates that this humoral immune response is unlikely to be the natural mechanism behind the HIV-resistance phenotype of persistently HIV-seronegative women. This result, however, does not preclude the further investigation of alloimmunization as an artificial HIV immunization strategy.

Creating CTL targets with epitope-linked beta 2-microglobulin constructs.

Eliciting a strong CTL response is dependent upon displaying suitably high levels of specific class I MHC/peptide complexes at the cell surface. In an effort to enhance the presentation of defined CTL target structures, two unique peptide-linked beta 2-microglobulin (beta 2m) molecules were constructed. The first, designated NP(366-374)-L8-h beta 2m, links the carboxyl terminus of the H-2Db-restricted influenza nucleoprotein (NP) epitope NP(366-374) to the amino terminus of h beta 2m through an eight-amino acid glycine/serine linker. The second molecule, designated NP(147-155)-L12-h beta 2m, similarly couples the H-2Kd-restricted influenza NP epitope NP(147-155) to h beta 2m via a 12-residue polypeptide linker. Transfection of the NP(366-374)-L8-h beta 2m vector into H-2b-expressing cell lines sensitized these cells for lysis by NP(366-374)-specific CTLs. Free NP peptide could not be detected when class I bound peptides were acid-extracted from the surface of NP(366-374)-L8-h beta2m transfectants, indicating that CTL killing was mediated by recognition of the peptide linked to h beta 2m and not by a degradation by-product. CTL target structure formation was also achieved by an exogenous presentation pathway. H-2d-expressing target cells were sensitized for lysis when pulsed with NP(147-155)-L12-h beta 2m protein derived from an Escherichia coli cell lysate. The effect of recombinant NP(147-155)-L12-h beta 2m was inhibited by competitor wild-type h beta 2m, indicating that the active peptide-h beta 2m fusion protein remained intact. The observation that beta 2m with covalently attached peptide can effectively create CTL target structures in vitro offers new possibilities for the in vivo induction of epitope-specific CTL responses by either DNA immunization or injection of the purified epitope-linked beta 2m.

Induction by DNA immunization of a protective antitumor cytotoxic T lymphocyte response against a minimal-epitope-expressing tumor.

In order to examine the use of DNA immunization to block tumor growth, we have developed a model system in which a defined 9-amino-acid epitope from the nucleoprotein of influenza virus is used as a surrogate tumor-associated antigen. A mastocytoma cell line of DBA/2 origin (P815) was transfected with a plasmid encoding the minimal H-2Kd-restricted NP(147-155) cytotoxic T lymphocyte (CTL) epitope, pCMV/NPep, to generate the cell line designated P815-NPep. Mice primed and boosted once with a plasmid encoding the full-length NP gene, pCMV/NP, but not with the minigene pCMV/NPep, developed a strong NP(147-155)-specific CTL response within 2 weeks after the boost. When challenged with 10(4) P815-NPep cells, pCMV/NP-immunized DBA/2 mice were protected from tumor challenge, whereas control mice immunized with the vector backbone rapidly developed lethal tumor. Importantly, the P815-NPep-immune mice were also protected from a subsequent challenge with the untransfected parental tumor P815. By depleting the NP-immune mice of either CD4+ or CD8+ T cells and then challenging with 10(4) P815-NPep tumor cells, it was determined that the CD8-depleted mice rapidly developed tumors, whereas the CD4-depleted or non-treated mice were protected. These data clearly indicate that intramuscular (i.m.) plasmid DNA immunization can be used to mobilize an effective CD8+ CTL-mediated antitumor response.

The immunotargeting approach to adjuvant-independent subunit vaccine design.

Recently there has been considerable interest in exploring adjuvant-independent approaches to the enhancement of immunogenicity. One strategy which has emerged in response to this need is the immunotargeting approach to subunit vaccine design. Immunotargeting involves the conjugation of non-self antigens to monoclonal antibodies specific for cell surface determinants (e.g. class II MHC) on antigen presenting cells in vivo. Saline injections of these immunoconjugates have been shown to promote significant IgG responses to the delivered antigens in a number of different animal model studies. Oral and nasal immunizations in mice with anti-class II MHC immunoconjugates induce both secretory IgA and systemic IgG responses. Recombinant anticlass II MHC antibodies with defined B-cell and T-cell epitopes incorporated into their primary sequence have recently been used to induce epitope specific antibody responses in macaques. Thus the potential now exists for the rational design of adjuvant-independent human vaccine candidates based on a recombinant immunotargeting anti-body framework.