Senior Member of the Staff
Head, Department of Cytokine Biology
email:
Medical School of Tringulo Mineiro, Uberaba, MG, Brazil, MD, 1968
Federal University of Rio de Janeiro, Brazil/Harvard Medical School, Ph.D., 1978, Immunology
University of So Paulo, Brazil, 1988, Professor of Microbiology and Immunology
Our laboratory focuses on research involving both systemic and oral infections. The World Health Organization estimates that tuberculosis, malaria, and HIV/AIDS, claims approximately 6 million lives every year, and cause debilitating illness in many millions more. These are the lives of infants, and children, and young adults in their most productive years. Unfortunately, there is still not an efficient vaccine for any of these three diseases. Yet, vaccines have proven to be the most effective means of controlling many other life-threatening infectious diseases. Over the past ten years global efforts have been directed toward the development of vaccines against tuberculosis, malaria, and HIV/AIDS as well as to several other serious infectious diseases. As part of this effort, our laboratory has focused on antigen discovery and vaccine development to tuberculosis and leishmaniasis. To achieve this goal we use modern molecular biology techniques combined with powerful immunological approaches. Thus far, an anti-leishmania vaccine has been developed and is currently in human trials and several promising candidates have been developed for diagnosing and preventing tuberculosis. More recently, after joining The Forsyth Institute, periodontal disease has been added as a major focus of interest of our laboratory.
To reach these goals our laboratory screens key pathogen antigens, using as readouts T cells from sensitized but healthy individuals or from animals immune to the diseases caused by these pathogens. Antigens that stimulate the appropriate subset of T cells (CD4+ Th1/Th2 T cells and CD8+ T cells) are selected for further studies, which involve cloning and expression of the genes encoding these antigens, followed by in vivo experiments in animal models aiming to understand the pathogenesis of the disease as well as to evaluate the vaccine potential of the cloned genes/antigens.
The current approach being used to identify antigens associated primarily with CD4+ T cell responses has been recently described by us in collaboration with scientists at the Infectious Disease Research Institute, Seattle, WA. Its general principle is based on the direct recognition by the T cells of antigens presented by macrophages or dendritic cells that have internalized a library of E. coli-containing expressed recombinant antigens. The library is initially divided into pools of approximately 50 transformants/well distributed in 96-well microtiter plates and stored in a replica plate manner. Macrophages or dendritic cells are fed with the E. coli, incubated for processing, washed and exposed to specific T cell lines in the presence of antibiotics to inhibit bacterial growth. T cell recognition is then detected by proliferation and cytokine production (IFN-gamma, IL-4, and IL-10). Wells that score positive are then broken down using the same protocol until a single clone is isolated. The gene is then sequenced, sub-cloned, expressed and the recombinant protein evaluated. This technology, can in principle detect microbial antigens that induce either CD4+ Th1 or Th2 responses, which is an important aspect of this technology; because such phenotypes of immune responses can in many circumstances be used as surrogates of T cells that mediate resistance or susceptibility to infection. For example, the Th1 response is protective against leishmaniasis while Th2 responses against this parasite are associated with susceptibility and aggravation of the disease. In contrast, Th2 responses are apparently protective against periodontal infection caused by Pophyromonas gingvalis while Th1 responses are associated with exacerbation of the disease.
CD8+ responses are also believed to play a role in resistance to tuberculosis and leishmaniasis and other infectious processes. To discover microbial antigens associated with CD8+ T cell responses, we use a direct approach. Peptides are isolated from MHC Class I molecules of infected cells, sequenced, and the sequences defined by bioinformatics as of pathogen origin are used to identify the genes to be cloned. This approach allows the direct cloning of pathogen genes encoding proteins that potentially are involved in the stimulation of anti-microbial CD8+ T cell immunity.
Following the steps related to discovery and production of recombinant proteins and plasmid DNA, prioritization of the antigens is based on their T cell recognition by peripheral blood mononuclear cells from healthy donors and by patients with active diseases as well as by T cells from lymphoid organs obtained from experimental models. Protection experiments are done in mice, guinea pigs, and monkeys after conventional immunization with the antigens mixed with various adjuvants or delivered in naked plasmid DNA format.
In addition to vaccine research, our laboratory is actively involved in the development of antigen detection assays for the diagnosis of both tuberculosis and leishmaniasis.
Kashino SS, Pollack N, Napolitano DR, Rodrigues V Jr, Campos-Neto A. 2008. Indentification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alterantive approach of antigen discovery of useful microbial molecules. Clin Exp Immunol. 153(1):56-62.
Leshem O, Kashino SS, Goncalves RB, Suzuki N, Onodera M, Fujimura A, Sasaki H, Stashenko P, Campos-Neto A. 2008. Th1 biased response to a novel Porphyromonas gingivalis protein aggravates bone resorption caused by this oral pathogen. Microbes Infect. 10(6):664-72.
Napolitano DR, Pollock N, Kashino SS, Rodrigues V Jr, Campos-Neto A. 2008. Identification of Mycobacterium tuberculosis ornithine carboamyltransferase in urine as a possible molecular marker of active pulmonary tuberculosis.Clin Vaccine Immunol. 15(4):638-43.
Stashenko P, Goncalves RB, Lipkin B, Ficarelli A, Sasaki H, Campos-Neto A. 2007. Th1 immune response promotes severe bone resorption caused by Porphyromonas gingivalis. Am J Pthol. 170(1):203-13.
Stashenko P, Goncalves R, Lipkin B, Sasaki H, Campos-Neto A. 2006. Inflammatory signaling in the dentin-pulp complex and periapical tissues. Adv. Dent. Res. In press
Kashino SS, Ovendale P, Izzo A, CamposNeto A. 2006. A unique model of dormant infection for vaccine development in tuberculosis. Clin. Vaccine Immunol. 13(9):1014–1021.
Gonçalves RB, Lesham O, Bernards K, Webb JR, Stashenko PP, CamposNeto A. 2006. T-cell expression cloning of Porphyromonas gingivalis genes coding for T helper-biased immune responses during infection. Infect. Immun. 74(7) :3958–3966.
Mukherjee S, Kashino SS, Zhang Y, Daifalla N, Rodrigues V. Jr, Reed SG, Campos-Neto A. 2005. Cloning of the gene encoding a protective Mycobacterium tuberculosis secreted protein detected in vivo during the initial phases of the infectious process. J. Immunol. 175(8):5298–5305.
Fujiwara RT, Vale AM, França Da Silva JC, Da Costa RT, Quetz J, Martins Filho OA, Reis AB, Corrêa Olivera R, Machado-Coelho GL, Bueno LL, Bethony JM, Frank G, Nascimento E, Genaro O, Mayrink W, Reed S, Campos-Neto A. 2005. Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis. Vet. Res. 36(5–6):827–838.
Campos-Neto A. 2005. What about Th1 /Th2 in cutaneous leishmaniasis vaccine discovery? Braz. J. Med. Biol. Res. 38(7):979–984.
Skeiky YAW, Alderson MR, Ovendale PJ, Lobet Y, Dalemans W, Orme IM, Reed SG, Campos-Neto A. 2005. Protection of mice and guinea pigs against tuberculosis induced by immunization with a single Mycobacterium tuberculosis recombinant antigen, MTB41. Vaccine 23(3):3937– 3945.
Reece ST, Stride S, Ovendale P, Reed SG, Campos-Neto A. 2005. Skin test performed with highly purified Mycobacterium tuberculosis recombinant protein triggers tuberculin shock in infected guinea pigs. Infect. Immun. 73(6):3301 –3306.
Liu C, Flamoe E, Chen H-J, Carter D, Reed SG, Campos-Neto A. 2004. Expression and purification of immunologically reactive DPPD, a recombinant Mycobacterium tuberculosis skin test antigen using Mycobacterium smegmatis and Escherichia coli host cells. Can. J. Microbiol. 50 (2) :97–1 05.
Campos-Neto A, Suffia I, Cavassani KA, Jen S, Greeson K, Ovendale P, Silva JS, Reed SG, Skeiky YA. 2003. Cloning and characterization of a gene encoding an immunoglobulin-binding receptor on the cell surface of some members of the family trypanosomatidae. Infect. Immun. 71(9) :5065–5076.
Reed SG, Campos-Neto A. 2003. Vaccines for parasitic and bacterial diseases. Curr. Opin. Immunol. 15(4) :456–460.
Reed SG, Coler RN, Campos-Neto A. 2003. Development of a leishmaniasia vaccine: The importance of MPL. Expert Rev. Vaccines 2(2):239–252.
Mukherjee S, Daifalla N, Liu C, Campos-Neto A. 2003. Alternative approach to express Mycobacterium tuberculosis proteins in Escherichia coli. Biotechniques 35(1):34–40.
Campos-Neto A, Webb JR, Greeson K, Coler RN, Skeiky YAW, Reed SG. 2002. Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice. Infect. Immun. 70(8):2828–2836.
Suely S.Kashino,Ph.D.
Onir Leshem, D.D.S
Danielle R. Napolitano, Ph.D.
