Kees L. M. C. Franken

Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses

One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNγ (P = 0.0003) and IL-5 (P < 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4+CD25+Foxp3+ T cells (P = 0.005) and displayed a lower HPV16-specific IFNγ/IL-10 ratio after vaccination (P < 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4+CD25+Foxp3+ T cells was predictive of clinical success. Foxp3+ T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.

Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase

Antigens of pathogenic microbes that mimic autoantigens are thought to be responsible for the activation of autoreactive T cells. Viral infections have been associated with the development of the neuroendocrine autoimmune diseases type 1 diabetes and stiff-man syndrome, but the mechanism is unknown. These diseases share glutamic acid decarboxylase (GAD65) as a major autoantigen. We screened synthetic peptide libraries dedicated to bind to HLA-DR3, which predisposes to both diseases, using clonal CD4+ T cells reactive to GAD65 isolated from a prediabetic stiff-man syndrome patient. Here we show that these GAD65-specific T cells crossreact with a peptide of the human cytomegalovirus (hCMV) major DNA-binding protein. This peptide was identified after database searching with a recognition pattern that had been deduced from the library studies. Furthermore, we showed that hCMV-derived epitope can be naturally processed by dendritic cells and recognized by GAD65 reactive T cells. Thus, hCMV may be involved in the loss of T cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity.

CD8+ CTL Priming by Exact Peptide Epitopes in Incomplete Freund’s Adjuvant Induces a Vanishing CTL Response, whereas Long Peptides Induce Sustained CTL Reactivity

Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8+ T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA257–264 peptide in IFA transiently activated CD8+ effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA323–339) restored both secondary expansion and in vivo effector functions of CD8+ T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8+ T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8+ T cell reactivity in vivo.

Immunogenicity of eight dormancy regulon-encoded proteins of Mycobacterium tuberculosis in DNA-vaccinated and tuberculosis-infected mice.

ABSTRACT Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one Kd-restricted T-cell epitope could be identified. BALB/c and (B6D2)F1 mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.

Immunogenicity of Novel DosR Regulon-Encoded Candidate Antigens of Mycobacterium tuberculosis in Three High-Burden Populations in Africa

ABSTRACT Increasing knowledge about DosR regulon-encoded proteins has led us to produce novel Mycobacterium tuberculosis antigens for immunogenicity testing in human populations in three countries in Africa to which tuberculosis (TB) is endemic. A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human immunodeficiency virus-negative adult household contacts of active pulmonary TB cases from South Africa (n = 56), The Gambia (n = 26), and Uganda (n = 49) were tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded M. tuberculosis recombinant protein antigens. ESAT-6/CFP10 fusion protein evoked responses in >75% of study participants in all three countries. Of the DosR regulon-encoded antigens tested, Rv1733c was the most commonly recognized by participants from both South Africa and Uganda and the third most commonly recognized antigen in The Gambia. The four most frequently recognized DosR regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c) included the three most immunogenic antigens in South Africa. In contrast, Rv3131 induced the highest percentage of responders in Gambian contacts (38%), compared to only 3.4% of Ugandan contacts and no South African contacts. Appreciable percentages of TB contacts with a high likelihood of latent M. tuberculosis infection responded to several novel DosR regulon-encoded M. tuberculosis proteins. In addition to significant similarities in antigen recognition profiles between the three African population groups, there were also disparities, which may stem from genetic differences between both pathogen and host populations. Our findings have implications for the selection of potential TB vaccine candidates and for determining biosignatures of latent M. tuberculosis infection, active TB disease, and protective immunity.

Superior induction of anti-tumor CTL immunity by extended peptide vaccines involves prolonged, DC-focused antigen presentation.

Anti‐tumor vaccines consisting of extended CTL peptides in combination with CpG‐ODN were shown to be superior to those comprising minimal CTL epitopes and CpG‐ODN, in that they elicit stronger effector CTL responses with greater tumoricidal potential. We now demonstrate that this improved performance is primarily due to the focusing of CTL epitope presentation onto activated DC in the inflamed lymph nodes draining the vaccination site. In the case of vaccination with minimal peptides, additional APC including T and B cells are also loaded with CTL epitopes. Our data suggest that circulation of these peptide‐loaded lymphocytes leads to epitope presentation in non‐inflamed lymphoid organs distal from the vaccination site, in the absence of potent costimulatory signals required for efficient CTL priming. The resulting blend of pro‐immunogenic and tolerogenic signals, which results in suboptimal activation of the CTL response, is avoided by vaccinating with extended CTL peptides. An additional advantage of extended CTL peptide vaccines is an increased duration of in vivo epitope presentation.

Lack of Immune Responses to Mycobacterium tuberculosis DosR Regulon Proteins following Mycobacterium bovis BCG Vaccination

ABSTRACT Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis (TB), despite its variable protective efficacy. Relatively little is known about the immune response profiles following BCG vaccination in relation to protection against TB. Here we tested whether BCG vaccination results in immune responses to DosR (Rv3133c) regulon-encoded proteins. These so-called TB latency antigens are targeted by the immune system during persistent Mycobacterium tuberculosis infection and have been associated with immunity against latent M. tuberculosis infection. In silico analysis of the DosR regulon in BCG and M. tuberculosis showed at least 97% amino acid sequence homology, with 41 out of 48 genes being identical. Transcriptional profiling of 14 different BCG strains, under hypoxia and nitric oxide exposure in vitro, revealed a functional DosR regulon similar to that observed in M. tuberculosis. Next, we assessed human immune responses to a series of immunodominant TB latency antigens and found that BCG vaccination fails to induce significant responses to latency antigens. Similar results were obtained with BCG-vaccinated BALB/c mice. In contrast, responses to latency antigens were observed in individuals with suspected exposure to TB (as indicated by positive gamma interferon responses to TB-specific antigens ESAT-6 and CFP-10) and in mice vaccinated with plasmid DNA encoding selected latency antigens. Since immune responses to TB latency antigens have been associated with control of latent M. tuberculosis infection, our findings support the development of vaccination strategies incorporating DosR regulon antigens to complement and improve the current BCG vaccine.

Response to Rv2628 latency antigen associates with cured tuberculosis and remote infection

Interferon-&ggr; release assays based on region of difference 1 antigens have improved diagnosis of latent tuberculosis infection (LTBI). However, these tests cannot discriminate between recently acquired infection (higher risk of progression to active tuberculosis) and remote LTBI. The objective of the present study was to evaluate the T-cell interferon-&ggr; responses to Mycobacterium tuberculosis DosR-regulon-encoded antigens (latency antigens) compared with QuantiFERON TB-Gold In-Tube (QFT-GIT) in subjects at different stages of tuberculosis. A total of 16 individuals with remote LTBI and 23 with recent infection were studied; 15 controls unexposed to M. tuberculosis and 50 patients with active tuberculosis and 45 with cured tuberculosis were also analysed. The results indicated that subjects with remote LTBI showed significantly higher whole-blood interferon-&ggr; responses to M. tuberculosis latency antigen Rv2628 than did individuals with recent infection, active tuberculosis and controls (p<0.003), whereas no significant differences between these groups were found for other latency antigens tested (Rv2626c, Rv2627c, Rv2031c and Rv2032). The proportion of responders to Rv2628 was five-fold higher among QFT-GIT-positive-individuals with remote infection than among those with recently acquired infection. These data suggest that responses to M. tuberculosis latency antigen Rv2628 may associate with immune-mediated protection against tuberculosis. In contact-tracing investigations, these preliminary data may differentiate recent (positive QFT-GIT results without responses to Rv2628) from remote infection (positive to both tests).

Epstein-Barr Virus gp42 Is Posttranslationally Modified To Produce Soluble gp42 That Mediates HLA Class II Immune Evasion

ABSTRACT Epstein-Barr virus (EBV) resides as a persistent infection in human leukocyte antigen (HLA) class II+ B lymphocytes and is associated with a number of malignancies. The EBV lytic-phase protein gp42 serves at least two functions: gp42 acts as the coreceptor for viral entry into B cells and hampers T-cell recognition via HLA class II molecules through steric hindrance of T-cell receptor-class II-peptide interactions. Here, we show that gp42 associates with class II molecules at their various stages of maturation, including immature αβIi heterotrimers and mature αβ-peptide complexes. When analyzing the biosynthesis and maturation of gp42 in cells stably expressing the viral protein, we found that gp42 occurs in two forms: a full-length type II membrane protein and a truncated soluble form. Soluble gp42 is generated by proteolytic cleavage in the endoplasmic reticulum and is secreted. Soluble gp42 is sufficient to inhibit HLA class II-restricted antigen presentation to T cells. In an almost pure population of Burkitts lymphoma cells in the EBV lytic cycle, both transmembrane and soluble forms of gp42 are detected. These results imply that soluble gp42 is generated during EBV lytic infection and could contribute to undetected virus production by mediating evasion from T-cell immunity.

T-Cell Responses to the Mycobacterium tuberculosis-Specific Antigen ESAT-6 in Brazilian Tuberculosis Patients

ABSTRACT The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-γ), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-γ secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-γ in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-γ in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-γ production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.