Specific Immunogenicity Research Articles

JJo, a Recombinant Dimer of Conformationally Restricted Peptide Elicits Protective Response against Group A Streptococcus (GAS) Isolates from a GAS-Endemic Region

A peptide (J14) containing conformationally restricted epitopes from the M protein of group A streptococcus (GAS) is capable of eliciting protective immune response against GAS infection. However, the protective response may be lost possibly due to its weak secondary-structure when the antigen is fused with other antigens in a recombinant polyepitope vaccine construct. We previously showed that JJo, a conformationally stabilized derivative of dimeric J14, overcomes this problem. We now show that anti JJo antibodies react with diverse GAS isolates found in the Indian sub-continent and that these antibodies are opsonic for GAS. The GAS strains used in this study were isolated from throat and skin swabs from Mumbai, Chennai and Vellore. Sera from mice immunized with recombinant JJo peptide were tested by ELISA, immunofluorescence, flow-cytometry, indirect bactericidal assay and mouse challenge assays to determine specific immunogenicity, opsonic functions and protection against an Indian isolate. We propose that JJo is a robust antigen suitable for inclusion in recombinant multi-epitope vaccines which are potentially affordable option for the pediatric population of developing countries.

Combining CpG and Al(OH)3 enhances the immunogenicity of hepatitis C virus recombinant ptotein combined vaccine

Objective To research CpG and Al(OH)3 adjuvants enhancing immunogenicity of hepatitis C virus(HCV) recombinant ptotein combined vaccine(TIE).Methods BALB/c mice were immunized with candidate vaccine TFE using CpG,Al(OH)3,Al(OH) 3 + CpG,or freund's adjuvant(FA) as the adjuvant.Five mice were sacrificed after 10 d of the last immunization.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Splenic cells were isolated and levels of IFN-γ,IL-4 and cytotoxic T lymphocyte(CTL) cytotoxicity assay were messuredin vitro.The remaining mice were subcutaneouly injected with 1 × 106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results The specific CTL activity of TFE + A1(OH) 3 + CpG group was higher than TFE + FA group and TFE + CpG group(P ＜ 0.05).The level of IFN-γsecreting cells in TFE + Al(OH)3 + CpG group was higher than that in TFE + M(OH)3 group or TFE + CpG group(P ＜ 0.05).Conclusion Combining Al(OH) 3 and CpG could enhance specific cellular immunogenicity of candidate HCV vaccine TFE.TFE + M(OH) 3 + CpG could effetively prevent the attack of tumor cell SP2/0-NS3 expressing nonstructural protein NS3 of HCV. Key words: Hepatitis C virus; Recombinant protein combined vaccine(TFE); Adjuvant; CpG; Al(OH)3

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma

BackgroundAnimal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. Patients and methodsWe report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). ResultsFull idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. ConclusionsThis manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.

Expression, purification and characterization of allelic variants of MSP-1 42 from Indian Plasmodium falciparum isolates

The C-terminal 19 and 42 kDa fragments of Plasmodium falciparum merozoite surface protein 1 (MSP-1) have shown to be protective in animals against lethal parasite challenge. The MSP-1 19 being highly conserved may lack sufficient number of T-cell epitopes in order to elicit a broader response in genetically diverse populations. The inclusion of additional epitopes from the N-terminal MSP-1 42 has shown to enhance the protective efficacy of MSP-1 19 vaccine. In an attempt to examine the strain specific immunogenicity to MSP-1, we have cloned and expressed three diverse allelic variants of MSP-1 42 from Indian P. falciparum isolates in bacteria. Among three alleles, one was extremely rare and not been found before. These purified and refolded recombinant products were recognized by conformation specific monoclonal antibodies and hyper-immune sera. Immunization of mice and rabbits with the purified proteins generated high titer biologically active polyclonal antibodies supporting further development of this vaccine candidate antigen.

Recombinant peptides as new immunogens for the control of the bovine tick, Rhipicephalus (Boophilus) microplus

A candidate vaccine that effectively controls Rhipicephalus microplus ticks in livestock is an attractive strategy in bovine management because of the expensive and labor-intensive treatment of animals with acaricides. However, the complex nature of ectoparasites has imposed restrictions on the development of such a vaccine, and its future efficacy is still under debate. Using Phage Display technology, we developed specific immunogens that mimic R. microplus tick antigens and were successfully validated by in vitro and in vivo assays. Nine peptides were used in the experimental vaccination of mice and cattle with different formulations. The peptides generated specific antibody response against R. microplus larval proteins in mouse immunization. A vaccine mixture of all nine selected phage clones was employed in cattle vaccination and the immune response induced specific changes in teleogine physiology, manifested as a hemorrhagic event in the gastrointestinal and reproductive tracts. A new means for vaccine development and the discovery of immunogens have been demonstrated, and our data may provide evidence that a putative vaccine for ectoparasite control is possible.

Haemagglutination assay of some human and animal arcobacter specie

Arcobacter are emerging food borne enteropathogens which can cause severe diarrhea and occasional systemic infection such as bacteraemia and peritonitis in humans. To understand the pathogenicity of this emerging pathogen, putative adhesive factors in the organism were studied by examining their ability to agglutinate erythrocytes from humans and other animal species. A total of 10 Arcobacter species from which four strains were obtained from humans and one from pigs in France. Five isolates were obtained from stool of healthy pigs and chicken in Nigeria. They were identified and confirmed by phenotypic and PCR techniques. Bacteria cells were washed in PBS by centrifugation. 20 μl of bacteria cells was reacted with equal volume of erythrocytes. Macroscopic haemagglutination was scored as either positive or negative after 5 minutes of mixing at room temperature. To understand the nature of red cell receptor for haemagglutinin, various sugars was used to inhibit previously positive haemagglutination reaction. Fresh cultures of all strains agglutinated erythrocytes from humans (blood group A+), sheep, guinea-pig and chicken. 20μl of all suspension containing 10 cells\ml gave a strong microscopic haemagglutination within 5 minutes after mixing with an equal volume of 3% erythrocytes suspension on a clean microscopic slide. No inhibition was observed with D glucose and mannose on previously positive haemagglutinating strains, but galactose inhibited by the same 50/50% (v|v). The result of this experiment provides a basis for further development of specific immunogen required for a more detailed study of characterization of specific adhesins. Keywords : Arcobacter, Haemagglutination, Adhesive factors, Nigeria

Induction of Tumor-specific Immune Response by Gene Transfer of Hsp70-cell-penetrating Peptide Fusion Protein to Tumors in Mice

To induce a tumor-specific immune response by delivering tumor-associated antigens in tumor cells to antigen-presenting cells (APCs), we designed a fusion protein which consists of heat-shock protein 70 (Hsp70) and the C-terminal 34 amino acids of herpes simplex virus VP22 protein (VP22(268-301)), the former having a peptide binding domain and an ability to be recognized by APCs, and the latter able to achieve cell penetration. Hsp70-VP22(268-301), the fusion protein, was efficiently taken up by mouse dendritic cell (DC) line DC2.4. Major histocompatibility complex (MHC) class I-restricted presentation of an epitope peptide of ovalbumin (OVA) was examined in DC2.4, and Hsp70-VP22(268-301) significantly increased the presentation of the peptide compared with Hsp70. Electroporation-assisted injection of naked plasmid vector expressing Hsp70-VP22(268-301) (pHsp70-VP22(268-301)) into subcutaneous tumors of EG7-OVA, a mouse lymphoma-expressing OVA, significantly increased the survival of mice compared with the same treatment with pHSp70, a plasmid expressing Hsp70. Splenocytes from the pHsp70-VP22(268-301)-treated mice exhibited cytolytic activity against both EG7-OVA and the parent EL4, but not against mouse melanoma B16-F10, suggesting that not only OVA-derived antigens but those common to EG7-OVA and EL4 are delivered to APCs. These results provide a new therapeutic method to induce tumor-specific antitumor immunity without identifying nor isolating tumor-associated antigens.

P19-32. HIV-1 vaccine design based on human vaccine trials to improve Gag, Pol, Nef and Env specific immune responses

Methods To improve Gag/Pol specific immunogenicity, some modifications were included comprising (i) re-introduction of the natural gag-pol frameshift allowing budding and release of GPN particles from transduced cells, (ii) inactivation the protease and (iii) separation of Gag (+G) and PolNef (PN) on two plasmids. Following various protocols, different combinations of GPN derivatives, G, PN and Env encoding plasmids were administered i.m. into Balb/c mice to overcome competition for presentation of relevant epitopes.

HLAMatchmaker-based definition of structural human leukocyte antigen epitopes detected by alloantibodies

This review addresses the concept that human leukocyte antigen (HLA) antibody specificity should be determined to HLA epitopes rather than HLA antigens. HLAMatchmaker is a computer algorithm that considers small configurations of polymorphic residues referred to as eplets as essential components of HLA epitopes. This overview describes recent developments that have increased our understanding of structural epitope antigenicity, that is, reactivity with specific antibody and immunogenicity, that is, its ability to induce an antibody response. A determination of the repertoire of immunogenic epitopes is important for HLA compatibility testing and the identification of acceptable mismatches for sensitized patients.

Delivery of Human Immunodeficiency Virus Vaccine Vectors to the Intestine Induces Enhanced Mucosal Cellular Immunity

Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4(+) T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low-pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon among all intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than after oral gavage, with rAd5 showing greater potency than the rAd35 or the rAd41 vector. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8(+) T-cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses.

The caveolin-1 binding domain of HIV-1 glycoprotein gp41 (CBD1) contains several overlapping neutralizing epitopes

The CBD1 peptide (SLEQI WNNMT WMQ WDK), corresponding to the consensus caveolin-1 binding domain in HIV-1 envelope glycoprotein gp41 (CBD1), elicits the production of antibodies that inhibit infection of primary CD4 + T lymphocytes by various primary HIV-1 isolates. Here we show that HIV-neutralizing antibodies against CBD1 react with multiple conformational epitopes that overlap the highly conserved caveolin-1 binding motif (CBM) with the N-terminal conserved isoleucine residue. The CBM-based peptides I WNNMT WMQ W and I WNNMT W when fused to a T helper epitope are immunogenic by inducing high titer CBM-specific antibodies capable of neutralizing HIV-1 infection in primary T lymphocyte cultures. Interestingly, neutralizing immune sera raised against a given peptide do not cross-react with related CBM-derived peptides, thus suggesting the existence of distinct neutralizing epitopes that probably reflect the dynamic conformational features of CBD1. In accord with this, the mixture of neutralizing immune sera raised against several CBM-derived peptides exerts a synergistic neutralizing activity against HIV-1 infection. Finally, the existence of several distinct overlapping epitopes in CBD1 is confirmed by murine monoclonal antibodies that we generated against the CBM-derived chimeric peptides. Our results indicate that CBD1- and CBM-based peptides mimic distinct dynamic conformations of CBD1, and thus such peptides could provide specific immunogens for an efficient vaccine preparation against HIV/AIDS infection.

Treatment of Biochemical Recurrence of Prostate Cancer With Granulocyte-Macrophage Colony-Stimulating Factor Secreting, Allogeneic, Cellular Immunotherapy

This phase I-II study evaluated the safety, clinical activity and immunogenicity of an immunotherapy developed from human prostate cancer cell lines (PC-3 and LNCaP) modified to secrete granulocyte-macrophage colony-stimulating factor. Patients with noncastrate prostate cancer (19) with biochemical (prostate specific antigen) recurrence following prostatectomy or radiation therapy and no radiological evidence of metastasis were enrolled in the study. Patients were injected with an initial dose of 5 x 10(8) cells followed by 12 biweekly administrations of 1 x 10(8) cells. The adverse event profile, prostate specific antigen response, changes in prostate specific antigen kinetics and immunogenicity were assessed. Immunotherapy was well tolerated with no serious treatment related adverse events and no autoimmune reactions. A negative deflection in prostate specific antigen slope was observed in 84% of patients after treatment with a significant increase in median prostate specific antigen doubling time from 28.7 weeks before treatment to 57.1 weeks after treatment (p = 0.0095). Median time to prostate specific antigen progression was 9.7 months. Immunoblot analysis of patient serum demonstrated new or enhanced production of PC-3 or LNCaP reactive antibodies in 15 of 19 (79%) patients after immunotherapy. Induction of antibody responses reactive against PC-3 in general, and to the PC-3 associated filamin-B protein specifically, were positively associated with treatment associated changes in prostate specific antigen kinetics. Granulocyte-macrophage colony-stimulating factor secreting cellular immunotherapy has a favorable toxicity profile with signals of clinical and immunological activity against hormone naïve prostate cancer. An association between immune response and prostate specific antigen changes was observed. Phase 3 trials in patients with advanced, metastatic, hormone refractory prostate cancer are under way.

Specific Immunogenicity of Heat Shock Protein gp96 Derives from Chaperoned Antigenic Peptides and Not from Contaminating Proteins

The peptide-binding property of MHC is central to adaptive immunological functions. A similar property of heat shock proteins (HSPs) hsp70 and hsp90 has been implicated in Ag presentation by MHC and in cross-priming. The peptide-binding pocket of hsp70 has been characterized structurally and functionally and a peptide-binding site in gp96 (of hsp90 family) has been defined. Nonetheless, questions persist whether the specific immunogenicity of HSP preparations derives from the peptides chaperoned by the HSPs or by proteins contaminating the HSP preparations. Because absolute purity of a protein preparation is a metaphysical concept, other approaches are necessary to address the question. In this study, we demonstrate that the specific immunogenicity of gp96 preparations isolated from cells expressing beta-galactosidase derives from the MHC I epitope precursors associated with the gp96 and not from contaminating beta-galactosidase protein nor unassociated fragments derived from it. Although the observations here are limited to a single HSP and antigenic peptides chaperoned by it, they can be extended broadly.

Spleen B cells from BALB/c are more prone to activation than spleen B cells from C57BL/6 mice during a secondary immune response to cruzipain

There is an increasing interest in the study of roles that B cells may play in regulating immune responses both in protection and in pathogenesis. However, little is known about additional immune functions of B cells independently of antibody production. In this study, we have assessed how the immunization with T-dependent antigens in different host genetic backgrounds affects several parameters of B cells during secondary immune responses. We have previously reported that BALB/c immunized with cruzipain, induced heart autoimmunity, whereas C57BL/6 mice were resistant. In a comparative study employing the same experimental model, we demonstrated that BALB/c-enriched spleen B cells presented higher ability to proliferate releasing elevated levels of IL-4. Moreover, spleen of immune BALB/c mice presented an increased number of germinal center and plasma cells as well as higher expression of B-cell activation markers (MHC class II, CD40, CD86). These findings demonstrate the influence of genetic background on B-cell activation and emphasize the importance of examining B-cell behavior in the context of the specific immunogens.

Suppression of Choroidal Neovascularization by Dendritic Cell Vaccination Targeting VEGFR2

To investigate whether the induction of cellular immunity against vascular endothelial growth factor receptor (VEGFR) 2 inhibits the development of choroidal neovascularization (CNV). H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGFR2 was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of the epitope peptide-pulsed mature DCs three times at 6-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, mice were killed to harvest the choroid and splenocytes. CNV volume was evaluated by volumetric measurements. To confirm the specific immunogenicity of the epitope peptides in C57BL/6 mice, CD8 T cells isolated from harvested splenocytes were restimulated to measure interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production through enzyme-linked immunospot assay and ELISA. To determine the T-cell subset responsible for the immunotherapy, mice were intraperitoneally injected with an anti-CD4 or anti-CD8 depletion antibody. CNV volume was significantly lower in mice immunized with the VEGFR2 epitope peptide than in those not immunized or immunized with a control peptide gp70. Cytokine assays showed the peptide-specific production of IFN-gamma and TNF-alpha from the CD8 T cells in a dose-dependent manner. In vivo depletion of CD8, but not CD4, T cells significantly reversed the suppressive effect of the VEGFR2 peptide-pulsed DC vaccination on CNV to the level observed in nonimmunized or gp70-immunized animals. These results indicate that the VEGFR2 peptide-specific induction of cellular immunity inhibits CNV through the cytotoxicity of CD8 T cells. Results of the present study suggested the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for CNV.

Immunohistochemical localization of a GHB receptor‐like protein isolated from rat brain

Gamma-hydroxybutyrate (GHB) is a substance derived from the metabolism of GABA and is heterogeneously distributed in various regions of the brain. This compound possesses a neuromodulatory role on several types of synapses, particularly those using GABA as a neurotransmitter. At physiological concentrations, this effect of GHB is mediated via specific receptors that induce neuronal hyperpolarization and bind radioactive GHB with a specific distribution, ontogenesis, kinetics, and pharmacology. A membrane protein that possesses six to seven transmembrane domains and which binds and is activated by micromolar amounts of GHB was recently cloned from rat brain hippocampus. In order to study the regional and cellular distribution of this receptor in rat brain, we selected several specific peptides belonging to the extracellular domains of the receptor to be used as specific immunogens to raise polyclonal antibodies in the rabbit. Among the antisera obtained, one of them gave particularly good results in terms of specificity and reactivity at high dilution. Immunohistochemical analyses, both at the confocal and electron microscopic level, showed receptor protein distribution closely resembling the distribution of GHB high-affinity binding sites, except for cerebellum, where GHB receptor(s) of lower affinity exist(s). In all regions studied the GHB receptor-like protein labeling appears to be distributed specifically in neurons and not in glial cells. At the cellular level the antibody specifically labels dendrites, and no immunoreactivity was detected in presynaptic endings or in axons. Accordingly, electron microscopy reveals strong labeling of postsynaptic densities and of neuronal cytosol.

Immunologic targeting: how to channel a minimal response for maximal outcome

Drugs that target extracellular molecules and intracellular pathways remain an area of active research in prostate cancer. Although preclinical data suggest that new drugs can modulate or slow prostate tumor proliferation, responses in man, such as disease stabilization or regression, are not as dramatic as those seen in preclinical models. Other approaches, including carbohydrate and cellular product vaccines, cytokines, and monoclonal antibodies either alone or with radiopharmaceuticals, are being used to seek and destroy cancer cells. Although robust in-vitro antibody responses can be generated against a specific immunogen in many vaccines, immunologists would agree that immune responses are suboptimal, as defined by a lack of impact on tumor growth, and insufficient to impact on disease progression. Although a preferred result, approaches that maximize the cellular arm of immune responses are limited by technology to detect these responses and by agents that can enhance their activity. DNA vaccines that target prostate-specific antigen and prostate-specific membrane antigen, and drugs that can block inhibitory molecules on T cells, such as cytotoxic T lymphocyte antigen-4, are currently under study. This article will review state-of-the-art mechanisms by which immunity may be enhanced to elicit antitumor responses against selectively expressed cell surface molecules and to maximize antitumor responses.

An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria

Many vaccines employed in childhood vaccination programmes are produced by conventional techniques, resulting in complex biological mixtures for which batch-related quality control requires in vivo potency testing. Monitoring consistency via in vitro tests during the vaccine production has the capacity to replace certain of the in vivo methods. In this respect, determining vaccine antigen immunogenicity through functional immunological tests has high potential. Advances in immunology have made it possible to analyse this biological activity by in vitro means. The present study established such an in vitro test system for tetanus toxoid (TT). This measured vaccine immunogenicity through an antigen-specific secondary (recall) response in vitro, using a porcine model growing in value for its closeness to human immune response characteristics. Discrimination between the specific recall TT antigen and diphtheria toxoid (DT) was possible using both peripheral blood mononuclear cell cultures and monocyte-derived dendritic cells in co-culture with autologous specific lymphocytes. TT-specific activation was detected with highest discrimination capacity using proliferation assays, as well as IFN-gamma and TT-specific antibody ELISPOTS (measuring secreting T and B lymphocytes, respectively). These in vitro systems show a high potential for replacing animal experimentation to evaluate the immunogenicity of complex vaccines.

A role for β-melanocyte-stimulating hormone in human body-weight regulation

Pro-opiomelanocortin (POMC) expressing neurons mediate the regulation of orexigenic drive by peripheral hormones such as leptin, cholecystokinin, ghrelin, and insulin. Most research effort has focused on alpha-melanocyte-stimulating hormone (alpha-MSH) as the predominant POMC-derived neuropeptide in the central regulation of human energy balance and body weight. Here we report a missense mutation within the coding region of the POMC-derived peptide beta-MSH (Y5C-beta-MSH) and its association with early-onset human obesity. In vitro and in vivo data as well as postmortem human brain studies indicate that the POMC-derived neuropeptide beta-MSH plays a critical role in the hypothalamic control of body weight in humans.

Dissection of ESAT-6 System 1 of Mycobacterium tuberculosis and Impact on Immunogenicity and Virulence

The dedicated secretion system ESX-1 of Mycobacterium tuberculosis encoded by the extended RD1 region (extRD1) assures export of the ESAT-6 protein and its partner, the 10-kDa culture filtrate protein CFP-10, and is missing from the vaccine strains M. bovis BCG and M. microti. Here, we systematically investigated the involvement of each individual ESX-1 gene in the secretion of both antigens, specific immunogenicity, and virulence. ESX-1-complemented BCG and M. microti strains were more efficiently engulfed by bone-marrow-derived macrophages than controls, and this may account for the enhanced in vivo growth of ESX-1-carrying strains. Inactivation of gene pe35 (Rv3872) impaired expression of CFP-10 and ESAT-6, suggesting a role in regulation. Genes Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877 encoding an ATP-dependent chaperone and translocon were essential for secretion of ESAT-6 and CFP-10 in contrast to ppe68 Rv3873 and Rv3876, whose inactivation did not impair secretion of ESAT-6. A strict correlation was found between ESAT-6 export and the generation of ESAT-6 specific T-cell responses in mice. Furthermore, ESAT-6 secretion and specific immunogenicity were almost always correlated with enhanced virulence in the SCID mouse model. Only loss of Rv3865 and part of Rv3866 did not affect ESAT-6 secretion or immunogenicity but led to attenuation. This suggests that Rv3865/66 represent a new virulence factor that is independent from ESAT-6 secretion. The present study has allowed us to identify new aspects of the extRD1 region of M. tuberculosis and to explore its role in the pathogenesis of tuberculosis.