Passive Transfer of Vaccine-Elicited Antibodies Protects against SIV in Rhesus Macaques.

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization.

Discovery and Selection of Hepatitis B Virus-Derived T Cell Epitopes for Global Immunotherapy Based on Viral Indispensability, Conservation, and HLA-Binding Strength.

Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies.

Effective Suppression of HIV-1 Replication by Cytotoxic T Lymphocytes Specific for Pol Epitopes in Conserved Mosaic Vaccine Immunogens.

Cytotoxic T lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize circulating HIV-1 could be key for both HIV-1 cure and prophylaxis. We recently designed conserved mosaic T-cell vaccine immunogens (tHIVconsvX) composed of 6 Gag and Pol regions. Since the tHIVconsvX vaccine targets conserved regions common to most global HIV-1 variants and employs a bivalent mosaic design, it is expected that it could be universal if the vaccine works. Although we recently demonstrated that CTLs specific for 5 Gag epitopes in the vaccine immunogens had strong ability to suppress HIV-1 replication in vitro and in vivo, it remains unknown whether the Pol region-specific CTLs are equally efficient. In this study, we investigated CTLs specific for Pol epitopes in the immunogens in treatment-naive Japanese patients infected with HIV-1 clade B. Overall, we mapped 20 reported and 5 novel Pol conserved epitopes in tHIVconsvX. Responses to 6 Pol epitopes were significantly associated with good clinical outcome, suggesting that CTLs specific for these 6 Pol epitopes had a strong ability to suppress HIV-1 replication in HIV-1-infected individuals. In vitro T-cell analyses further confirmed that the Pol-specific CTLs could effectively suppress HIV-1 replication. The present study thus demonstrated that the Pol regions of the vaccine contained protective epitopes. T-cell responses to the previous 5 Gag and present 6 Pol protective epitopes together also showed a strong correlation with better clinical outcome. These findings support the testing of the conserved mosaic vaccine in HIV-1 cure and prevention in humans.IMPORTANCE It is likely necessary for an effective AIDS vaccine to elicit CD8+ T cells with the ability to recognize circulating HIV-1 and suppress its replication. We recently developed novel bivalent mosaic T-cell vaccine immunogens composed of conserved regions of the Gag and Pol proteins matched to at least 80% globally circulating HIV-1 isolates. Nevertheless, it remains to be proven if vaccination with these immunogens can elicit T cells with the ability to suppress HIV-1 replication. It is well known that Gag-specific T cells can suppress HIV-1 replication more effectively than T cells specific for epitopes in other proteins. We recently identified 5 protective Gag epitopes in the vaccine immunogens. In this study, we identified T cells specific for 6 Pol epitopes present in the immunogens with strong abilities to suppress HIV-1 in vivo and in vitro This study further encourages clinical testing of the conserved mosaic T-cell vaccine in HIV-1 prevention and cure.

CD4+ T Cells Are Not Required for Suppression of Hepatitis B Virus Replication in the Liver of Vaccinated Chimpanzees.

Humans vaccinated with hepatitis B virus (HBV) surface antigen (HBsAg) sometimes develop humoral and cellular immunity to HBV proteins such as core and polymerase that are not vaccine components, providing indirect evidence that vaccine-induced immunity is not sterilizing. We previously described CD4(+) T-cell immunity against HBsAg and polymerase in chimpanzees after vaccination and HBV challenge. Here, vaccinated chimpanzees with protective levels of anti-HBsAg antibodies were rechallenged with HBV after antibody-mediated CD4(+) T-cell depletion. HBV DNA was detected in liver for at least 3 months after rechallenge, but virus replication was suppressed, as revealed by the absence of HBV DNA and HBsAg in serum. These observations provide direct virological evidence for nonsterilizing immunity in individuals with anti-HBsAg antibodies and are consistent with translation of HBV proteins to prime immune responses. They also indicate that CD4(+) T cells were not required for suppression of HBV replication in previously vaccinated individuals.

Mucosal priming with a replicating-vaccinia virus-based vaccine elicits protective immunity to simian immunodeficiency virus challenge in rhesus monkeys.

Mucosal surfaces are not targeted by most human immunodeficiency virus type 1 (HIV-1) vaccines, despite being major routes for HIV-1 transmission. Here we report a novel vaccination regimen consisting of a mucosal prime with a modified replicating vaccinia virus Tiantan strain (MVTT(SIVgpe)) and an intramuscular boost with a nonreplicating adenovirus strain (Ad5(SIVgpe)). This regimen elicited robust cellular immune responses with enhanced magnitudes, sustainability, and polyfunctionality, as well as higher titers of neutralizing antibodies against the simian immunodeficiency virus SIV(mac1A11) in rhesus monkeys. The reductions in peak and set-point viral loads were significant in most animals, with one other animal being protected fully from high-dose intrarectal inoculation of SIV(mac239). Furthermore, the animals vaccinated with this regimen were healthy, while ~75% of control animals developed simian AIDS. The protective effects correlated with the vaccine-elicited SIV-specific CD8(+) T cell responses against Gag and Pol. Our study provides a novel strategy for developing an HIV-1 vaccine by using the combination of a replicating vector and mucosal priming.

Frequent detection of herpes simplex virus antigen in skin and peripheral blood CD34+ mononuclear cells from patients with graft-versus-host disease.

Viruses are implicated in the initiation or flare of graft-versus-host disease (GVHD) by virtue of their ability to activate antigen-presenting dendritic cells (DC). Herpes simplex virus (HSV) infects circulating CD34+ stem cell progenitors, favoring their differentiation into skin homing DC (CD1a+ Langerhans cells) that contribute to the development of an inflammatory skin rash known as HSV-associated erythema multiforme (HAEM). Following on these findings, we conducted a prospective study to examine whether HSV is also associated with GVHD. Skin biopsies and peripheral blood mononuclear cells (PBMC) were collected from 37 consecutive patients on admission before and after allogeneic hematopoietic stem cell transplantation (HSCT) and examined for HSV antigen (Pol) expression and the presence of Pol+CD34+ and Pol+CD1a+ cells. Sixteen patients developed a skin rash that was histopathologically consistent with GVHD (group I), 3 patients had a rash that was not GVHD (group II, EM-like) and 18 patients did not develop any rash after HSCT (group III). Skin biopsies from the group I patients were Pol negative pre-HSCT (baseline) but became Pol+ after the diagnosis of GVHD. The GVHD biopsies also contained Pol+CD34+ and Pol+CD1a+ cells, and these patients had a significant percentage of circulating Pol+CD34+ and Pol+CD1a+ PBMC. By contrast, the group II patients had Pol+ skin cells and Pol+CD34+ circulating PBMC at baseline that decreased post-HSCT. The group III patients had Pol negative skin and very few circulating Pol+CD34+ and Pol+CD1a+ PBMC at baseline that were not significantly changed post-HSCT. The data associate skin GVHD with HSV reactivation during conditioning and its propensity for nonreplicative infection of CD34+ PBMC that induces DC activation. Further studies are needed to better elucidate this association.

GagPol-specific CD4⁺ T-cells increase the antibody response to Env by intrastructural help.

BACKGROUND: Immunization of rhesus macaques against Gag of SIV resulted in a more rapid appearance of Env antibodies after infection with SIV or SHIV challenge viruses although the vaccines lacked an Env component. We therefore explored whether T helper cells specific for internal HIV proteins could provide intrastructural help for Env-specific B cells and thus increase the Env antibody response. RESULTS: Mice were immunized by adenoviral vector or DNA vaccines against GagPol and then boosted with virus-like particles (VLP) containing GagPol and Env. Env-specific antibody levels after the VLP booster immunizations were significantly higher in GagPol-immunized mice than in mock-vaccinated controls. Adoptive transfer of CD4+ T cells from GagPol-immunized mice also enhanced the Env antibody response to VLP immunization in the recipient mice. Depending on the presence of VLPs, co-cultivation of CD4+ T cells from GagPol-primed mice with BCR transgenic B cells specific for a protein presented on the surface of the VLPs also resulted in the activation of the B and T cells. CONCLUSIONS: Our study indicates that GagPol-specific T helper cells may provide intrastructural help for Env antibody responses. This cross-talk between immune responses directed against different components of the retroviral particle may be relevant for the immunopathogenesis of retroviral infections and allow to improve virus like particle vaccine approaches against HIV.

Circumventing antivector immunity by using adenovirus-infected blood cells for repeated application of adenovirus-vectored vaccines: proof of concept in rhesus macaques.

Adenovirus has been extensively exploited as a vector platform for delivering vaccines. However, preexisting antiadenovirus immunity is the major stumbling block for application of adenovirus-vectored vaccines. In this study, we found that freshly isolated peripheral blood mononuclear cells (PBMCs), mostly CD14(+) cells, from adenovirus serotype 5 (Ad5)-seropositive primates (humans and rhesus macaques) can be efficiently infected with Ad5 in vitro. On the basis of this observation, a novel strategy based on adenoviral vector-infected PBMC (AVIP) immunization was explored to circumvent antivector immunity. Autologous infusion of Ad5-SIVgag-infected PBMCs elicited a strong Gag-specific cellular immune response but induced weaker Ad5-neutralizing antibody (NAb) in Ad5-seronegative macaques than in macaques intramuscularly injected with Ad5-SIVgag. Moreover, Ad5-seropositive macaques receiving multiple AVIP immunizations with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines elicited escalated Env-, Gag-, and Pol-specific immune responses after each immunization that were significantly greater than those in macaques intramuscularly injected with these Ad5-SIV vaccines. After challenged intravenously with a highly pathogenic SIVmac239 virus, macaques receiving AVIP immunization demonstrated a significant reduction in viral load at both the peak time and set-point period compared with macaques without Ad5-SIV vaccines. Our study warranted further research and development of the AVIP immunization as a platform for repeated applications of adenovirus-vectored vaccines.

HIV-DNA priming alters T cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable.

Many candidate HIV vaccines are designed to primarily elicit T cell responses. Although repeated immunization with the same vaccine boosts Ab responses, the benefit for T cell responses is ill defined. We compared two immunization regimens that include the same recombinant adenoviral serotype 5 (rAd5) boost. Repeated homologous rAd5 immunization fails to increase T cell responses, but increases gp140 Ab responses 10-fold. DNA prime, as compared with rAd5 prime, directs long-term memory CD8(+) T cells toward a terminally differentiated effector memory phenotype with cytotoxic potential. Based on the kinetics of activated cells measured directly ex vivo, the DNA vaccination primes for both CD4(+) and CD8(+) T cells, despite the lack of detection of the latter until after the boost. These results suggest that heterologous prime-boost combinations have distinct immunological advantages over homologous prime-boosts and suggest that the effect of DNA on subsequent boosting may not be easily detectable directly after the DNA vaccination.

PAComplex: a web server to infer peptide antigen families and binding models from TCR-pMHC complexes.

One of the most adaptive immune responses is triggered by specific T-cell receptors (TCR) binding to peptide-major histocompatibility complexes (pMHC). Despite the availability of many prediction servers to identify peptides binding to MHC, these servers are often lacking in peptide-TCR interactions and detailed atomic interacting models. PAComplex is the first web server investigating both pMHC and peptide-TCR interfaces to infer peptide antigens and homologous peptide antigens of a query. This server first identifies significantly similar TCR-pMHC templates (joint Z-value ≥ 4.0) of the query by using antibody-antigen and protein-protein interacting scoring matrices for peptide-TCR and pMHC interfaces, respectively. PAComplex then identifies the homologous peptide antigens of these hit templates from complete pathogen genome databases (≥10(8) peptide candidates from 864,628 protein sequences of 389 pathogens) and experimental peptide databases (80,057 peptides in 2287 species). Finally, the server outputs peptide antigens and homologous peptide antigens of the query and displays detailed interacting models (e.g. hydrogen bonds and steric interactions in two interfaces) of hitTCR-pMHC templates. Experimental results demonstrate that the proposed server can achieve high prediction accuracy and offer potential peptide antigens across pathogens. We believe that the server is able to provide valuable insights for the peptide vaccine and MHC restriction. The PAComplex sever is available at http://PAcomplex.life.nctu.edu.tw.

Hepatitis B virus polymerase blocks pattern recognition receptor signaling via interaction with DDX3: implications for immune evasion.

Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF) activation and ultimately interferon (IFN) production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV), which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol) blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1)/IkappaB kinase-epsilon (IKKepsilon), the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKepsilon activity by disrupting the interaction between IKKepsilon and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKepsilon activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.

Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.

We aimed to determine the prevalence of the coexistence of HBsAg and anti-HBs and to analyze the clinical and virological features of infection, including amino acid (aa) patterns of the S gene and reverse transcriptase (RT) region in Chinese chronic hepatitis B (CHB) patients. Fifty-four (2.90%) CHB patients who were positive for both HBsAg and anti-HBs were tested, and sequences were obtained from 52 of them as well as 48 patients from a control group. S gene and RT region sequences were amplified and sequenced using in-house protocols. There was no significant difference between patients with and without anti-HBs with regard to age, gender, alanine aminotransferase level, and the proportion positive for HBeAg and HBcAb. The occurrence of genotype C (P = 0.001) and anti-HBeAb positivity (P = 0.027) was significantly higher in HBsAg+/anti-HBs+ individuals. In the S gene, the number of mutated residues in the HBsAg+/anti-HBs+ group was markedly higher than in control patients (1.88 versus 1.02 substitutions per 100 amino acids, P = 0.022). The amino acid exchange occurred mostly within the N-terminal region (2.15 versus 0.87 substitutions per 100 amino acids, P = 0.023) and the "a" determinant (3.61 versus 1.56 substitutions per 100 amino acids, P = 0.049) in the two groups. In the RT region, the mean number of substitution per 100 aa showed a tendency to be significantly higher in HBsAg+/anti-HBs+ patients than in controls (2.34 versus 1.46, P = 0.040). This study showed a prevalence of coexistence of anti-HBs in HBsAg-positive patients and an increased frequency of genotype C and aa variability within both HBsAg and RT involving functionally important regions of those proteins.

Robust vaccine-elicited cellular immune responses in breast milk following systemic simian immunodeficiency virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys.

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection: differential CTL responses to HIV-1 and Epstein-Barr virus in late disease.

Major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to human persistent virus infections. Measurements of the frequency and specificity of human immunodeficiency virus type 1 (HIV-1)-specific CTL and their variation with time may indicate their relative importance in modulating the progression of HIV-1 infection. We have used limiting dilution analysis (LDA) to derive quantitative estimates of the frequency of HIV-1-specific CTL precursors in a cross-sectional study of 23 patients at different clinical stages of HIV-1 infection and to compare these with the frequency of CTL precursors specific for another persistent virus (Epstein-Barr virus [EBV]) in the same patients. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with autologous HIV-1-infected lymphoblasts and assayed for cytotoxicity in 51Cr release assays against autologous and MHC-mismatched lymphoblastoid B cells infected with recombinant vaccinia viruses expressing the three HIV-1 structural gene products. The frequency of MHC-restricted precursors was high in asymptomatic HIV-1-infected patients (env-specific CTL precursors up to 73/10(6) PBMC; gag-specific CTL precursors up to 488/10(6) PBMC), although the relative frequency against the different structural gene products varied from patient to patient. The HIV-1-specific CTL precursor frequency was reduced in patients who had more severe (< 400/microliters) CD4+ lymphocyte depletion, while in the majority of such patients the frequency of CTL precursors against EBV was maintained at levels observed in healthy controls. Direct CTL activity in unstimulated PBMC was observed in three of nine patients but no correlation was found between the presence of an activated CTL response and the magnitude of the CTL response detected after stimulation in LDA. Thus, CTL precursors were detected against all three HIV-1 structural gene products in patients with CD4+ lymphocyte counts > 400/microliters, at frequencies that are high compared with those reported for other persistent viruses. A CTL response directed against multiple protein antigens of HIV-1 may protect the patient against epitope variation. The fact that the EBV-specific CTL precursor frequencies were maintained in advanced HIV-1 infection suggests that there may be selective impairment of the HIV-1-specific CTL response associated with disease progression.

Evidence of widespread binding of HLA class I molecules to peptides.

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.

Patterns of immunodominance in HIV-1-specific cytotoxic T lymphocyte responses in two human histocompatibility leukocyte antigens (HLA)-identical siblings with HLA-A*0201 are influenced by epitope mutation.

Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201-restricted SLYNTVATL and HLA-A3-restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201-restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response.

Structural features of peptide analogs of human histocompatibility leukocyte antigen class I epitopes that are more potent and immunogenic than wild-type peptide.

Certain peptide analogs that carry substitutions at residues other than the main major histocompatibility complex anchors and are surprisingly much more antigenic than wild-type peptide (heteroclitic analogs). To date, it was unknown how frequently wild-type epitopes could be modified to obtain heteroclitic activity. In this study, we analyzed a large panel of analogs of two different human histocompatibility leukocyte antigen (HLA)-A2.1-restricted epitopes and found that heteroclitic analogs were associated with higher magnitude responses and increased (up to 10(7)-fold) sensitivity to antigen, and corresponded to conservative or semiconservative substitutions at odd-numbered positions in the middle of the peptide (positions 3, 5, or 7). These findings were validated by performing additional immunogenicity studies in murine and human systems with four additional epitopes. The biological relevance of heteroclitic analogs was underlined when predicted analogs of the p53.261 epitope was shown to induce cytotoxic T lymphocytes (CTLs) that recognize low concentrations of peptide (high avidity) in vivo and demonstrate in vitro antitumor recognition. Finally, in vitro immunization of human peripheral blood mononuclear cells with two heteroclitic analogs resulted in recruitment of more numerous CTLs which were associated with increased antigen sensitivity. In conclusion, heteroclitic analogs were identified in each of the six cases studied and structural features were defined which allow identification of such analogs. The strong CTL immunity elicited by heteroclitic epitopes suggest that they could be of significant value in vaccination against tolerant or weakly immunogenic tumor-associated and viral antigens.

Amelioration of colitis by genetically engineered murine regulatory T cells redirected by antigen-specific chimeric receptor.

BACKGROUND & AIMS: The therapeutic application of regulatory T cells (Tregs) for the treatment of inflammatory diseases is limited by the scarcity of antigen-specific Tregs. A preferred approach to endow effector T cells (Teff) with a desired specificity uses chimeric immune receptors with antibody-type specificity. Accordingly, employing such chimeric immune receptors to redirect Tregs to sites of inflammation may be a useful therapeutic approach to alleviate a broad scope of diseases in which an uncontrolled inflammatory response plays a major role. METHODS: To enable application of the approach in clinical setting, which requires the genetic modification of the patient's own Tregs, we describe here a novel protocol that allows the efficient retroviral transduction and 2,4,6-trinitrophenol-specific expansion of murine naturally occurring regulatory T cells (nTregs), with a 2,4,6-trinitrophenol-specific tripartite chimeric receptor. RESULTS: Transduced Tregs maintained their Foxp3 level, could undergo repeated expansion upon ex vivo encounter with their cognate antigen in a major histocompatibility complex-independent, costimulation-independent, and contact-dependent manner and specifically suppressed Teff cells. Adoptive transfer of small numbers of the transduced nTregs was associated with antigen-specific, dose-dependent amelioration of trinitrobenzenesulphonic acid colitis. CONCLUSIONS: This study demonstrates that nTregs can be efficiently transduced to express functional, antigen-specific chimeric receptors that enable the specific suppression of effector T cells both in vitro and in vivo. This approach may enable future cell-based therapeutic application in inflammatory bowel disease, as well as other inflammatory disorders.

T-cell vaccination reduces simian immunodeficiency virus levels in semen.

Recent findings suggest that most sexual transmission of human immunodeficiency virus type 1 (HIV-1) occurs during the acute phase of infection when viral replication is most intense. Here, we show that vaccine-elicited cellular immune responses can significantly reduce simian immunodeficiency virus levels in the semen during the period of primary infection in monkeys. A vaccine that decreases the quantity of HIV-1 in the semen of males during primary infection might decrease HIV-1 transmission in human populations and therefore affect the spread of AIDS.