RESUMO
We previously identified potential correlates of vaccine-induced protection against Francisella tularensis using murine splenocytes and further demonstrated that the relative levels of gene expression varied significantly between tissues. In contrast to splenocytes, peripheral blood leukocytes (PBLs) represent a means to bridge vaccine efficacy in animal models to that in humans. Here we take advantage of this easily accessible source of immune cells to investigate cell-mediated immune responses against tularemia, whose sporadic incidence makes clinical trials of vaccines difficult. Using PBLs from mice vaccinated with F. tularensis Live Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitro Francisella replication within macrophages with gene expression analyses. The in vitro functions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivo protection conferred by LVS-derived vaccines. Moreover, several genes previously identified by the evaluation of splenocytes were also found to be differentially expressed in immune PBLs. In addition, more extensive screening identified additional potential correlates of protection. Finally, expression of selected genes in mouse PBLs obtained shortly after vaccination, without ex vivo restimulation, was different among vaccine groups, suggesting a potential tool to monitor efficacious vaccine-induced immune responses against F. tularensis. Our studies demonstrate that murine PBLs can be used productively to identify potential correlates of protection against F. tularensis and to expand and refine a comprehensive set of protective correlates.
Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Linfócitos/metabolismo , Tularemia/prevenção & controle , Animais , Técnicas de Cocultura , Regulação da Expressão Gênica , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise Serial de Proteínas , Proteínas/genética , Proteínas/metabolismo , Baço/citologiaRESUMO
In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rß2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.
Assuntos
Vacinas Bacterianas , Biomarcadores/metabolismo , Francisella tularensis/imunologia , Tularemia/prevenção & controle , Animais , Vacinas Bacterianas/normas , Técnicas de Cocultura , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Francisella tularensis/genética , Francisella tularensis/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/genética , Estimativa de Kaplan-Meier , Linfócitos/citologia , Linfócitos/imunologia , Macrófagos/citologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Tularemia/imunologia , Tularemia/microbiologia , Regulação para Cima/genética , Vacinas Atenuadas/normasRESUMO
We examined the relationship between the association of a vaccine antigen with immune cells in secondary lymphoid organs shortly after immunization and the resulting neutralizing antibody response induced by that antigen using three antigenic forms of anthrax protective antigen (PA) that induce qualitatively different antibody responses. The three PA forms used were wild-type PA, which binds to anthrax toxin receptors and elicits a robust antibody response that includes both neutralizing and non-neutralizing antibodies; a receptor-binding-deficient (RBD) mutant form of PA, which does not bind cellular receptors and elicits only barely detectable antibody responses; and an engineered chimeric form of PA, which binds cholera toxin receptors and elicits a robust total antibody response but a poor neutralizing antibody response. We found that both wild-type PA and the PA chimera associated with immune cells in secondary lymphoid organs after immunization, but the RBD mutant PA exhibited minimal association, revealing a relationship between antigen binding to toxin receptors on immune cells after immunization and subsequent antibody responses. A portion of wild-type PA that bound to immune cells was cell surface-associated and maintained its native conformation. Much lower amounts of conformationally intact PA chimera were associated with immune cells after immunization, correlating with the lower neutralizing antibody response elicited by the PA chimera. Thus, binding of an antigen to receptors on immune cells in secondary lymphoid organs after immunization and maintenance of conformational integrity of the cell-associated antigen help dictate the magnitude of the resulting neutralizing antibody response, but not necessarily the total antibody response.IMPORTANCEMany vaccines protect by the induction of antibodies that neutralize the action of the pathogen. Here, we followed the fate of three antigenic forms of a vaccine antigen in secondary lymphoid organs after immunization to investigate events leading to a robust neutralizing antibody response. We found that the magnitude of the neutralizing antibody response, but not the total antibody response, correlates with the levels of conformationally intact antigen associated with immune cells in secondary lymphoid organs after primary immunization. We believe that these results provide important insights into the genesis of neutralizing antibody responses induced by vaccine antigens and may have implications for vaccine design.
Assuntos
Vacinas contra Antraz , Bacillus anthracis , Anticorpos Neutralizantes , Formação de Anticorpos , Antígenos de Bactérias/metabolismo , Vacinação , Imunização , Anticorpos Antibacterianos , Bacillus anthracis/metabolismoRESUMO
Staphylococcus aureus is one of the most common etiological agents of community-acquired skin and soft tissue infection (SSTI). Although the majority of S. aureus community-acquired SSTIs are uncomplicated and self-clearing in nature, some percentage of these cases progress into life-threatening invasive infections. Current animal models of S. aureus SSTI suffer from two drawbacks: these models are a better representation of hospital-acquired SSTI than community-acquired SSTI, and they involve methods that are difficult to replicate. For these reasons, we sought to develop a murine model of community-acquired methicillin-resistant S. aureus SSTI (CA-MRSA SSTI) that can be consistently reproduced with a high degree of precision. We utilized this model to begin to characterize the host immune response to this type of infection. We infected mice via epicutaneous challenge of the skin on the outer ear pinna using Morrow-Brown allergy test needles coated in S. aureus USA300. When mice were challenged in this model, they developed small, purulent, self-clearing lesions with predictable areas of inflammation that mimicked a human infection. CFU in the ear pinna peaked at day 7 before dropping by day 14. The T(h)1 and T(h)17 cytokines gamma interferon (IFN-γ), interleukin-12 (IL-12) p70, tumor necrosis factor alpha (TNF-α), IL-17A, IL-6, and IL-21 were all significantly increased in the draining lymph node of infected mice, and there was neutrophil recruitment to the infection site. In vivo neutrophil depletion demonstrated that neutrophils play a protective role in preventing bacterial dissemination and fatal invasive infection.
Assuntos
Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/patologia , Modelos Animais de Doenças , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/patologia , Animais , Carga Bacteriana , Citocinas/análise , Citocinas/imunologia , Orelha Externa/microbiologia , Orelha Externa/patologia , Feminino , Linfonodos/química , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Pele/microbiologia , Pele/patologia , Fatores de TempoRESUMO
IL-12p40 plays an important role in F. tularensis Live Vaccine Strain (LVS) clearance that is independent of its functions as a part of the heterodimeric cytokines IL-12p70 or IL-23. In contrast to WT, p35, or p19 knockout (KO) mice, p40 KO mice infected with LVS develop a chronic infection that does not resolve. Here, we further evaluated the role of IL-12p40 in F. tularensis clearance. Despite reduced IFN-γ production, primed splenocytes from p40 KO and p35 KO mice appeared functionally similar to those from WT mice during in vitro co-culture assays of intramacrophage bacterial growth control. Gene expression analysis revealed a subset of genes that were upregulated in re-stimulated WT and p35 KO splenocytes, but not p40 KO splenocytes, and thus are candidates for involvement in F. tularensis clearance. To directly evaluate a potential mechanism for p40 in F. tularensis clearance, we reconstituted protein levels in LVS-infected p40 KO mice using either intermittent injections of p40 homodimer (p80) or treatment with a p40-producing lentivirus construct. Although both delivery strategies yielded readily detectable levels of p40 in sera and spleens, neither treatment had a measurable impact on LVS clearance by p40 KO mice. Taken together, these studies demonstrate that clearance of F. tularensis infection depends on p40, but p40 monomers and/or dimers alone are not sufficient.
Assuntos
Subunidade p40 da Interleucina-12 , Tularemia , Animais , Camundongos , Vacinas Bacterianas , Citocinas/metabolismo , Francisella tularensis , Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tularemia/imunologiaRESUMO
Identifying suitable animal models and standardizing preclinical methods are important for the generation, characterization, and development of new vaccines, including those against Francisella tularensis. Non-human primates represent an important animal model to evaluate tularemia vaccine efficacy, and the use of correlates of vaccine-induced protection may facilitate bridging immune responses from non-human primates to people. However, among small animals, Fischer 344 rats represent a valuable resource for initial studies to evaluate immune responses, to identify correlates of protection, and to screen novel vaccines. In this study, we performed a comparative analysis of three Fischer rat substrains to determine potential differences in immune responses, to evaluate methods used to quantify potential correlates of protection, and to evaluate protection after vaccination. To this end, we took advantage of data previously generated using one of the rat substrains by evaluating two live vaccines, LVS and F. tularensis SchuS4-ΔclpB (ΔclpB). We compared immune responses after primary vaccination, adaptive immune responses upon re-stimulation of leukocytes in vitro, and sensitivity to aerosol challenge. Despite some detectable differences, the results highlight the similarity of immune responses to tularemia vaccines and challenge outcomes between the three substrains, indicating that all offer acceptable and comparable approaches as animal models to study Francisella infection and immunity.
RESUMO
Francisella tularensis, the causative agent of tularemia, is classified as Tier 1 Select Agent with bioterrorism potential. The efficacy of the only available vaccine, LVS, is uncertain and it is not licensed in the U.S. Previously, by using an approach generally applicable to intracellular pathogens, we identified working correlates that predict successful vaccination in rodents. Here, we applied these correlates to evaluate a panel of SchuS4-derived live attenuated vaccines, namely SchuS4-ΔclpB, ΔclpB-ΔfupA, ΔclpB-ΔcapB, and ΔclpB-ΔwbtC. We combined in vitro co-cultures to quantify rodent T-cell functions and multivariate regression analyses to predict relative vaccine strength. The predictions were tested by rat vaccination and challenge studies, which demonstrated a clear relationship between the hierarchy of in vitro measurements and in vivo vaccine protection. Thus, these studies demonstrated the potential power a panel of correlates to screen and predict the efficacy of Francisella vaccine candidates, and in vivo studies in Fischer 344 rats confirmed that SchuS4-ΔclpB and ΔclpB-ΔcapB may be better vaccine candidates than LVS.
RESUMO
CCR2 is the major chemokine receptor that regulates appropriate trafficking of inflammatory monocytes, but the role of this chemokine receptor and its ligands during primary and secondary infection with intracellular infections remains incompletely understood. Here we used murine infection with the Live Vaccine Strain (LVS) of Francisella tularensis to evaluate the role of CCR2 during primary and secondary parenteral responses to this prototype intracellular bacterium. We find that mice deficient in CCR2 are highly compromised in their ability to survive intradermal infection with LVS, indicating the importance of this receptor during primary parenteral responses. Interestingly, this defect could not be readily attributed to the activities of the known murine CCR2 ligands MCP-1/CCL2, MCP-3/CCL7, or MCP-5/CCL12. Nonetheless, CCR2 knockout mice vaccinated by infection with low doses of LVS generated optimal T cell responses that controlled the intramacrophage replication of Francisella, and LVS-immune CCR2 knockout mice survived maximal lethal Francisella challenge. Thus, fully protective adaptive immune memory responses to this intracellular bacterium can be readily generated in the absence of CCR2.
Assuntos
Francisella tularensis/fisiologia , Receptores CCR2/genética , Tularemia/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL7/deficiência , Quimiocina CCL7/genética , Quimiocina CCL7/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Francisella tularensis/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/metabolismo , Receptores CCR2/deficiência , Taxa de Sobrevida , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tularemia/mortalidade , Tularemia/patologia , Tularemia/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologiaRESUMO
Inhalation of small numbers of Francisella tularensis subspecies tularensis (Ftt) in the form of small particle aerosols causes severe morbidity and mortality in people and many animal species. For this reason, Ftt was developed into a bona fide biological weapon by the USA, by the former USSR, and their respective allies during the previous century. Although such weapons were never deployed, the 9/11 attack quickly followed by the Amerithrax attack led the U.S. government to seek novel countermeasures against a select group of pathogens, including Ftt. Between 2005-2009, we pursued a novel live vaccine against Ftt by deleting putative virulence genes from a fully virulent strain of the pathogen, SCHU S4. These mutants were screened in a mouse model, in which the vaccine candidates were first administered intradermally (ID) to determine their degree of attenuation. Subsequently, mice that survived a high dose ID inoculation were challenged by aerosol or intranasally (IN) with virulent strains of Ftt. We used the current unlicensed live vaccine strain (LVS), first discovered over 70 years ago, as a comparator in the same model. After screening 60 mutants, we found only one, SCHU S4 ΔclpB, that outperformed LVS in the mouse ID vaccination-respiratory-challenge model. Currently, SCHU S4 ΔclpB has been manufactured under current good manufacturing practice conditions, and tested for safety and efficacy in mice, rats, and macaques. The steps necessary for advancing SCHU S4 ΔclpB to this late stage of development are detailed herein. These include developing a body of data supporting the attenuation of SCHU S4 ΔclpB to a degree sufficient for removal from the U.S. Select Agent list and for human use; optimizing SCHU S4 ΔclpB vaccine production, scale up, and long-term storage; and developing appropriate quality control testing approaches.
RESUMO
Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as a prototype. We compared rM-CSF with L929 supernatant in terms of their effects on mouse and rat macrophage growth, maturation patterns, surface marker expression, and the expression of selected genes. Further, we compared macrophage infectivity and bacterial replication using LVS. Finally, we compared the in vitro function of BMDMs co-cultured with splenocytes from vaccinated animals in terms of their control of intramacrophage bacterial replication, as well as production of cytokines and nitric oxide. We demonstrated that rM-CSF produced BMDMs with similar, or minimal, phenotypic and gene expression outcomes compared to those generated with media containing L929 supernatant. Most importantly, functional outcomes were similar. Taken together, our data support the use of the rM-CSF in cell culture media as an alternative to L929-supplemented media for functional bioassays that use C57BL/6J mouse or Fischer 344 rat BMDMs to study intracellular infections. This comparison therefore facilitates future protocol standardization.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Infecções Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Bioensaio/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura/métodos , Feminino , Fibroblastos , Francisella tularensis/imunologia , Regulação da Expressão Gênica/imunologia , Imunoensaio/métodos , Linfócitos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/metabolismo , Vacinas Atenuadas/imunologiaRESUMO
Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naïve and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium.
Assuntos
Interferon gama/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Francisella tularensis/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Neutrófilos/metabolismo , Baço/metabolismo , Linfócitos T/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/imunologia , Tularemia/microbiologiaRESUMO
Parenteral and respiratory vaccinations with the intracellular bacterium Francisella tularensis have been studied using the live vaccine strain (LVS) in a mouse model, and spleen cells from immune mice are often used for immunological studies. However, mechanisms of host immunological responses may be different in nonlymphoid organs that are important sites of infection, such as lung and liver. Using parenteral (intradermal) or respiratory (cloud aerosol) vaccination, here we examine the functions of resulting LVS-immune liver or lung cells, respectively. Surprisingly, LVS was considerably more virulent when administered by cloud aerosol than by intranasal instillation, suggesting method-dependent differences in initial localization and/or dissemination patterns. Only low doses were sublethal, and resolution of sublethal cloud aerosol infection was dependent on gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible nitric oxide synthase. Nonetheless, survival of cloud aerosol or parenteral infection resulted in the development of a protective immune response against lethal LVS intraperitoneal or aerosol challenge, reflecting development of systemic secondary immunity in both cases. Such immunity was further detected by directly examining the functions of LVS-immune lung or liver lymphocytes in vitro. Lung lymphocytes primed by respiratory infection, as well as liver lymphocytes primed by parenteral infection, clearly controlled in vitro intracellular bacterial growth primarily via mechanisms that were not dependent on IFN-gamma activity. Thus, our results indicate functional similarities between immune T cells residing in spleens, livers, and lungs of LVS-immune mice.
Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Fígado/imunologia , Pulmão/imunologia , Linfócitos T/imunologia , Tularemia/prevenção & controle , Animais , Contagem de Colônia Microbiana , Feminino , Francisella tularensis/crescimento & desenvolvimento , Interferon gama/deficiência , Interferon gama/imunologia , Fígado/microbiologia , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/microbiologia , Análise de SobrevidaRESUMO
We demonstrated previously that mice treated with bacterial or oligonucleotide DNA containing unmethylated CpG motifs are transiently protected against lethal parenteral challenge with the intracellular bacterium Francisella tularensis Live Vaccine Strain (LVS). Here we explore the cellular basis of this protection. Wild-type mice that were treated with CpG oligonucleotide DNA and challenged with a lethal dose of LVS survived, while mice lacking TLR9 did not. In vitro, treatment of LVS-infected macrophages and/or naive splenocytes with oligo DNA had no impact on intracellular bacterial replication. In contrast, in vitro co-culture of LVS-infected macrophages with splenocytes obtained from mice treated with oligo DNA in vivo resulted in control of intracellular LVS growth. Control was reversed by antibodies to interferon-gamma or to tumor necrosis factor-alpha and by inhibition of nitric oxide, and to a lesser degree by antibodies to Interleukin-12. Further, splenocytes from DNA-primed normal, T cell KO, B cell KO, lymphocyte-deficient scid, or perforin KO mice all controlled intra-macrophage LVS growth. Enriched DNA-primed natural killer cells, but not B cells, clearly controlled intracellular LVS growth. Thus, NK cells contribute to DNA-mediated protection by production of cytokines including IFN-gamma and TNF-alpha, resulting in nitric oxide production and control of intracellular Francisella replication.
Assuntos
DNA Bacteriano/imunologia , Francisella tularensis/crescimento & desenvolvimento , Células Matadoras Naturais/imunologia , Tularemia/imunologia , Animais , Ilhas de CpG/imunologia , DNA Bacteriano/farmacologia , Francisella tularensis/imunologia , Francisella tularensis/patogenicidade , Interferon gama/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Óxido Nítrico/biossíntese , Oligonucleotídeos/imunologia , Tularemia/microbiologia , Fator de Necrose Tumoral alfa/biossíntese , VirulênciaRESUMO
Francisella tularensis, a small gram-negative intracellular bacterium responsible for causing tularemia, is highly pathogenic and classified as a category A agent of bioterrorism. As for other intracellular pathogens, successful protective immune responses to Francisella tularensis require rapid and efficient induction of gamma interferon (IFN-gamma) production. Studies using intracellular bacteria such as Listeria monocytogenes as well as Francisella suggest that natural killer (NK) and T cells are important sources of IFN-gamma. However, comprehensive characterization of specific sources of IFN-gamma produced during Francisella infection in vivo remains incomplete, and depletion of NK cells before infection of mice with the F. tularensis live vaccine strain (LVS) has little impact on the course or outcome of infection. In this study, we determined the cell subpopulations that respond quickly to intradermal F. tularensis LVS infection of mice by producing IFN-gamma within hours to a few days. Splenic and liver lymphocytes were obtained from LVS-infected mice and analyzed for IFN-gamma mRNA by reverse transcription-PCR, for intracellular cytokine expression by multiparameter flow cytometry, and for ex vivo production of IFN-gamma protein by enzyme-linked immunosorbent assay. Cells producing IFN-gamma were readily detectable by day 3 after infection, and numbers progressively increased through days 5 to 7. Importantly, the cell types responsible for IFN-gamma production were much more varied than expected: these included not only NK cells and T cells, which might be predicted, but also other cells, including dendritic cells (DCs), "NK DCs," NK T cells, and neutrophils. Most importantly, since RAG-1 knockout mice appeared to exhibit a frequency of IFN-gamma-producing cells comparable to that of intact wild-type mice, early IFN-gamma production by innate immune cells does not depend on the presence of T or B cells.
Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Interferon gama/biossíntese , Linfócitos/imunologia , Células Mieloides/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Injeções Intradérmicas , Células Matadoras Naturais/imunologia , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Linfócitos T/imunologiaRESUMO
There are no defined correlates of protection for any intracellular pathogen, including the bacterium Francisella tularensis, which causes tularemia. Evaluating vaccine efficacy against sporadic diseases like tularemia using field trials is problematic, and therefore alternative strategies to test vaccine candidates like the Francisella Live Vaccine Strain (LVS), such as testing in animals and applying correlate measurements, are needed. Recently, we described a promising correlate strategy that predicted the degree of vaccine-induced protection in mice given parenteral challenges, primarily when using an attenuated Francisella strain. Here, we demonstrate that using peripheral blood lymphocytes (PBLs) in this approach predicts LVS-mediated protection against respiratory challenge of Fischer 344 rats with fully virulent F. tularensis, with exceptional sensitivity and specificity. Rats were vaccinated with a panel of LVS-derived vaccines and subsequently given lethal respiratory challenges with Type A F. tularensis. In parallel, PBLs from vaccinated rats were evaluated for their functional ability to control intramacrophage Francisella growth in in vitro co-culture assays. PBLs recovered from co-cultures were also evaluated for relative gene expression using a large panel of genes identified in murine studies. In vitro control of LVS intramacrophage replication reflected the hierarchy of protection. Further, despite variability between individuals, 22 genes were significantly more up-regulated in PBLs from rats vaccinated with LVS compared to those from rats vaccinated with the variant LVS-R or heat-killed LVS, which were poorly protective. These genes included IFN-γ, IL-21, NOS2, LTA, T-bet, IL-12rß2, and CCL5. Most importantly, combining quantifications of intramacrophage growth control with 5-7 gene expression levels using multivariate analyses discriminated protected from non-protected individuals with greater than 95% sensitivity and specificity. The results therefore support translation of this approach to non-human primates and people to evaluate new vaccines against Francisella and other intracellular pathogens.
Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Francisella tularensis/patogenicidade , Imunização , Sistema Respiratório/microbiologia , Animais , Feminino , Regulação da Expressão Gênica/imunologia , Imunidade Humoral/imunologia , Macrófagos/microbiologia , Análise Multivariada , Ratos , Linfócitos T/imunologia , VirulênciaRESUMO
Human carcinoembryonic antigen (CEA) is an oncofetal glycoprotein overexpression of which by gastrointestinal carcinomas is well known. Expression of CEA in head and neck cancer (HNC) is not widely recognized. It is important to note that most of these studies used polyclonal antibodies that may have cross-reactivity with CEA-related antigens. Currently, CEA is being evaluated in preclinical and clinical studies as a target for specific immunotherapy against gastrointestinal adenocarcinomas that express the antigen. This study was conducted to evaluate CEA as a potential target for specific immunotherapy against HNC. Immunohistochemical analysis of tumor tissue from 69 cases of squamous cell carcinoma (SCC) of the head and neck using a CEA-specific monoclonal antibody (COL-1) showed the majority to be positive for CEA. Tumor cell lines derived from human HNC were screened for CEA transcripts using nested reverse transcription-PCR. Constitutive expression of CEA mRNA was detected in 7 of 10 HNC lines. CEA protein was detectable in lysates from all 7 of the lines by quantitative fluoroimmunometry. SDS-PAGE/Western blot analysis of cell lysates from these lines showed a COL-1 immunoreactive product with a molecular weight equivalent to that of CEA. Cell surface expression of CEA was low for the SCC lines; however, there was moderate to strong cytoplasmic staining intensity for all of the CEA(+) HNC lines by immunocytochemistry. Additional supportive evidence for CEA as a target was demonstrated by the presence of cytolytic activity of an HLA-A2-restricted/CEA-epitope-specific human CTL against a CEA-overexpressing HNC-derived SCC line. These results suggest that CEA may be considered as a possible target for specific vaccine-mediated immunotherapy against HNCs.
Assuntos
Antígeno Carcinoembrionário/imunologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/terapia , Imunoterapia Adotiva/métodos , Antígeno Carcinoembrionário/biossíntese , Carcinoma de Células Escamosas/metabolismo , Antígeno HLA-A2/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Imuno-Histoquímica , Linfócitos T Citotóxicos/imunologiaRESUMO
Renewed interest in Francisella tularensis has resulted in substantial new information about its pathogenesis and immunology, along with development of useful animal models. While understanding of protective immunity against Francisella remains incomplete, data in both animals and humans suggest that inducing T cell-mediated immunity is crucial for successful vaccination with current candidates such as the Live Vaccine Strain (LVS), with specific antibodies and immune B cells playing supporting roles. Consistent with this idea, recent results indicate that measurements of T cell functions and relative gene expression by immune T cells predict vaccine-induced protection in animal models. Because field trials of new vaccines will be difficult to design, using such measurements to derive potential correlates of protection may be important to bridge between animal efficacy studies and people.
Assuntos
Vacinas Bacterianas/imunologia , Vacinas Bacterianas/isolamento & purificação , Francisella tularensis/imunologia , Tularemia/prevenção & controle , Animais , Biomarcadores/análise , Modelos Animais de Doenças , Francisella tularensis/patogenicidade , Humanos , Linfócitos T/imunologia , Tularemia/imunologiaRESUMO
Our laboratory has employed in vitro and in vivo mouse models based on Francisella tularensis Live Vaccine Strain (LVS)-induced protection to elucidate immune correlates for intracellular bacteria. Among the effectors found was GM-CSF, a pleiotropic cytokine that is integral to the development and proliferation of myeloid cells, including alveolar macrophages. GM-CSF has roles in resistance to primary murine infection with several intracellular pathogens, but its role during Francisella infection is unknown. Francisella is an intracellular pathogen that infects lungs after inhalation, primarily invading alveolar macrophages. Here we show that GM-CSF has route-dependent roles during primary infection of mice with LVS. GM-CSF deficient (GM-CSF KO) mice were slightly more susceptible than wild type to intradermal infection, but had increased resistance to intranasal infection. Similarly, these mice had increased resistance to pulmonary infection with virulent F. tularensis (SchuS4). LVS-vaccinated GM-CSF KO mice had normal adaptive immune responses, as measured by T cell activities after LVS intradermal or intranasal vaccination, and survived lethal secondary LVS challenge. GM-CSF KO mice also had robust humoral responses, producing elevated levels of serum antibodies following LVS vaccination compared to wild type mice. Taken together, our data demonstrates that the absence of GM-CSF improves resistance to pulmonary, but not intradermal, infection with Francisella.
Assuntos
Francisella tularensis/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunidade nas Mucosas , Mucosa Nasal/imunologia , Pele/imunologia , Tularemia/imunologia , Animais , Anticorpos Antibacterianos/sangue , Modelos Animais de Doenças , Imunidade Celular , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologiaRESUMO
PURPOSE: HuCC49V10 (V10), a humanized anticarcinoma monoclonal antibody (Ab) CC49, was generated by grafting only the specificity-determining regions (SDRs) of CC49 onto the variable light and variable heavy frameworks of the human Abs LEN and 21/28'CL, respectively. SDRs are those residues of the complementarity-determining regions that are most critical for antigen (Ag) binding. Compared with HuCC49, which was developed by conventional complementarity-determining region grafting, V10 has lower reactivity to the sera from patients who were previously given murine CC49 in clinical trials, although its Ag-binding affinity is 2-3-fold lower than that of HuCC49. To generate variants of V10 with higher Ag-binding affinity and lower sera reactivity, in vitro affinity maturation of V10 was carried out using phage display technique. EXPERIMENTAL DESIGN: A limited library of Fabs was generated by replacing some of the SDRs with all possible residues located at the corresponding positions in human Abs. The library was enriched, by several rounds of panning, in Fabs that have high affinity for the TAG-72 Ag. The clones encoding the best binders were expressed in insect cells as whole Abs that were purified and characterized. RESULTS: Competition radioimmunoassay and surface plasmon resonance measurements showed that two of the isolates, V14 and V15, have higher binding affinity than that of V10. In addition, the surface plasmon resonance analysis showed that the variants V14 and V15, compared with the parental V10, have lower reactivity to the anti-V region Abs using sera from patients who received murine CC49. CONCLUSIONS: The two isolates, V14 and V15, which show higher Ag-binding reactivity and lower sera reactivity than the parental V10 Ab, are potentially more useful clinical reagents. These results demonstrate that phage display can be used to isolate variants of an Ab that are potentially less immunogenic in patients than the parental Ab from which they are derived.
Assuntos
Adenocarcinoma/imunologia , Anticorpos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Substituição de Aminoácidos , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/uso terapêutico , Complexo Antígeno-Anticorpo , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Sequência de Bases , Primers do DNA , Desenho de Fármacos , Amplificação de Genes , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Mutagênese Sítio-Dirigida , Relação Estrutura-AtividadeRESUMO
The murine mAb CC49 specifically recognizes a tumor-associated glycoprotein (TAG)-72, which is expressed on the majority of human carcinomas. This Ab has potential applications in the diagnosis and treatment of human carcinomas. However, patients receiving murine CC49 generate human anti-murine Ab (HAMA) responses, preventing repeated administration of the Ab for effective treatment. To minimize the HAMA response, two versions of humanized CC49 (HuCC49) were developed: (a) HuCC49 and (b) HuCC49V10 (V10). HuCC49 was developed by grafting the CC49 CDRs, while V10 was generated by grafting only the specificity determining residues (SDRs) of the CC49 onto the frameworks of the human Abs. During the generation of both HuCC49 and V10, a few murine framework residues that were believed to be essential for the integrity of the Ag-binding site were retained. However, the indispensability of these residues for the Ag-binding activity of CC49 has not been experimentally validated. In this study, an array of V10 variants were generated by replacing, by site-specific mutagenesis, the murine framework residues that were retained in the humanized Ab with their counterparts in the human templates. The variants were tested for their (a) Ag-binding activity and (b) reactivity to sera from patients who were previously administered murine CC49 in a clinical trial. One such variant, V59, compared to the parental V10, shows a significant decrease in its reactivity to the anti-variable region Abs present in the patients' sera, while it binds to the TAG-72 Ag with a slightly higher affinity. Variant 59, which is expected to be minimally immunogenic because of its low sera reactivity, is a potentially useful clinical reagent against human carcinomas. In this study, we show for the first time that experimental validation rather than reliance on the protein data bank (PDB) should be the criterion for the indispensability of framework residues for the humanization of any murine Ab to retain its Ag-binding property and reduce its immunogenicity in patients.