Malaria and other vector-borne infection surveillance in the U.S. Department of Defense Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance program: review of 2009 accomplishments

Vector-borne infections (VBI) are defined as infectious diseases transmitted by the bite or mechanical transfer of arthropod vectors. They constitute a significant proportion of the global infectious disease burden. United States (U.S.) Department of Defense (DoD) personnel are especially vulnerable to VBIs due to occupational contact with arthropod vectors, immunological naiveté to previously unencountered pathogens, and limited diagnostic and treatment options available in the austere and unstable environments sometimes associated with military operations. In addition to the risk uniquely encountered by military populations, other factors have driven the worldwide emergence of VBIs. Unprecedented levels of global travel, tourism and trade, and blurred lines of demarcation between zoonotic VBI reservoirs and human populations increase vector exposure. Urban growth in previously undeveloped regions and perturbations in global weather patterns also contribute to the rise of VBIs. The Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) and its partners at DoD overseas laboratories form a network to better characterize the nature, emergence and growth of VBIs globally. In 2009 the network tested 19,730 specimens from 25 sites for Plasmodium species and malaria drug resistance phenotypes and nearly another 10,000 samples to determine the etiologies of non-Plasmodium species VBIs from regions spanning from Oceania to Africa, South America, and northeast, south and Southeast Asia. This review describes recent VBI-related epidemiological studies conducted by AFHSC-GEIS partner laboratories within the OCONUS DoD laboratory network emphasizing their impact on human populations

Human Lymphocyte Engraftment and Function in HU-PBL-SCID Mice: a Dissertation

The immune system is responsible for defending a host animal from a wide variety of threats. Manipulation of the immune system can result in beneficial outcomes such as immunity to pathogens, or deleterious outcomes such as autoimmunity. Advances in our understanding of how the immune system develops and functions have benefited greatly from studies in animals, particularly in mice where the genetics are well known and a multitude of reagents are readily available for experimental use. Although much has been learned from animal experimentation, it must be cautioned that animals are not humans. Unforeseen outcomes and complications often arise when translating research obtained in animal models to treatment of human patients. A small animal model in which the human immune system can be established and manipulated experimentally in vivo would be valuable for the study of human immune responses in infectious diseases, transplantation, and autoimmunity, and ultimately for translation of these findings to the human patient. Contribution to this model is the overall goal of this thesis. The severe combined immunodeficiency (Prkdcscid, termed scid) mutation was discovered in 1983 in the C.B-17 strain of mice. Scid mice lack functional T and B lymphocytes and are unable to mount immune responses or reject allogeneic or xenogeneIc grafts. However, C.B-17-scid mice do develop normal or even elevated innate immune function. Based on the ability of scid mice to accept xenografts, human peripheral blood mononuclear cells have been injected to generate Hu-PBL-scid mice. These Hu-PBL-scid mice have been proposed as an in vivo model of the human immune system. Although this model was described over 12 years ago, there are a number of obstacles that impede its ability to recapitulate human immunity. The Hu-PBL-scid mouse model established using C.B-17-scid mice as recipients is hindered by 1) low levels of human leukocyte engraftment, 2) engraftment of predominately memory/activated human T cells with specificity directed against host murine MHC antigens, 3) rapid transition of engrafted human lymphocytes to a functionally anergic state, and 4) a paucity of knowledge providing an understanding of the mechanisms that underlie engraftment and function of the human lymphocytes in the immunodeficient hosts. This thesis was initiated at the time the NOD-scid mouse became available for establishment of Hu-PBL-scid mice, and this model has subsequently been termed Hu-PBL-NOD-scid. The NOD-scid mouse was designed as an improved recipient for human PBL based on its innate immune characteristics. Defects in innate immunity in wild-type NOD/Lt mice include reduced NK cell activity, defects in macrophage development and function, and a lack of hemolytic complement. NOD-scid mice retained these characteristics, and engraft human cells at higher levels compared to C.B-17-scid mice. However, the ratio of CD4+ to CD8+ T cells in Hu-PBL-NOD-scid mice remained skewed towards the CD8+ population, similar to that of the Hu-PBL-C.B-17-scid mouse. My thesis was based on the overall hypothesis that additional genetic manipulation of the recipient strain would enhance further the engraftment and function of human cells. Based on the engraftment results obtained in NOD-scid mice, we performed these genetic manipulations using the NOD-scid strain as the reference. We first hypothesized that removal of MHC expression would lower human anti-mouse xenoreactivity and enhance engraftment of naïve T cells, a cell population not readily detectable in Hu-PBL-C.B-17-scid or Hu-PBL-NOD-scid mice. Towards this goal, the NOD-scid mouse expressing a targeted mutation for beta-2 microglobulin (B2mnull) mouse was created by Dr. Leonard Shultz at the Jackson Laboratory. Because B2m is required for expression of MHC class I and for development of functional NK cells, we predicted that NOD-scid-B2mnull mice would first exhibit a normalized CD4:CD8 T cell ratio resulting from reduced CD8 engraftment due to decreased human anti-mouse MHC class I reactivity. Since MHC class I molecule expression is required for the development of NK cells, we further predicted that there would be a reduction of NK cell activity, permitting enhanced engraftment. Data presented in this thesis demonstrates that human PBL engraft in NOD-scid-B2mnull mice at levels higher than NOD-scid mice, and with an increased CD4:CD8 T cell ratio. The mechanism(s) responsible for the increased engraftment of human cells in these mice became a major focus of this thesis. This thesis is composed of three Specific Aims. Specific Aim 1 was to determine the mechanism(s) underlying human cell engraftment and function in Hu-PBL-scid mice. These data are contained in Part 1 of the Results. Specific Aim 2 was to elucidate the costimulation interactions between human T cells and murine host APCs that control the level of engraftment and activation state of the human lymphocytes. These data are contained in Part 2 of the Results. Specific Aim 3 was to utilize these fundamental observations to initiate studies into the induction of primary human immune responses in Hu-PBL-scid mice. Although the goal of these latter studies was not attained, the data from the experiments performed is provided in Part 3 of the Results, and is discussed relative to future directions for this arm of the project. In Part 1 of the Results, we utilized in vitro and in vivo techniques to study the underlying mechanisms regulating human cell engraftment and function in scid mice. We observed that the absence of mouse MHC class I antigens in NOD-scid-B2mnull mice does not reduce the stimulatory capacity of APCs toward human T cells in vitro. We further demonstrated that naïve human T cells persist for at least 2 weeks in the peritoneal cavity of Hu-PBL-NOD-scid and Hu-PBL-NOD-scid-B2mnull mice. However, in both strains of recipients, the human cells progress to an anergic phenotype as documented by cell surface molecule expression and by functional activity. We further documented that the increased engraftment of human CD4+ T cells observed in NOD-scid-B2mnull mice is due predominantly to the ablation of host NK cell activity. Increased engraftment of human CD4+ cells in NOD-scid-B2mnull mice can be recapitulated in NOD-scid mice by antibody-mediated depletion of residual host NK cells. Finally, we demonstrated that expression of MHC class II molecules by recipient mice facilitated stimulation and engraftment of human cells. However, mouse MHC class II expression is not required for human cell engraftment into scid mice. In Part 2 of the Results, we addressed the cellular and costimulatory interactions of engrafted human cells in scid mice. Costimulatory interactions between T cells and APC are now known to be critical for the proper activation and functioning of cells in the immune system. Understanding the role of costimulatory interactions between human T cells, human APCs, and mouse APCs became a major focus of this thesis. We demonstrated that human CD4+ T cells are required for the engraftment of human CD8+ T cells. The mechanism by which human CD4+ cells mediate this helper activity requires expression of CD154. Antibody-mediated blockade of CD154 in vivo abrogates human cell engraftment in scid mice. The role of host APCs in the engraftment of human lymphocytes was demonstrated by blocking host CD40 with antibody. Preventing human T cell CD154 interaction with host CD40 on murine APCs blocked human cell engraftment in scid mice, demonstrating the importance of trans-costimulation in human T cell activation. This trans-costimulation appeared to be mediated by B7 expression on mouse APCs. We further demonstrated that in vivo depletion of human CD8+ T cells in Hu-PBL-NOD-scid mice leads to increased levels of human CD4+ T cells, elevated human immunoglobulin in the serum, and increased incidence of EBV-related lymphoproliferative disorders. These observations suggested that human CD8+ T cells are able to regulate human CD4+ T cell help and provide surveillance activity for EBV-related lymphoproliferative disorders. In Part 3 of the Results, we used the Hu-PBL-scid mouse to initiate experiments designed to generate primary human immune responses in vivo. These experiments were based on our observation that few if any human APCs survive in scid mice, and on reports that dendritic cells (DC) are required for activation of naïve T cells and initiation of a primary immune response. We used recombinant viruses expressing HIV-1 proteins that are being developed as potential vaccines for HIV as our immunizing reagent. For APCs, we used DCs from NOD-scid mice expressing human MHC class II molecules as the source of APCs presenting antigen to the engrafted human T cells in the scid mice. Our attempts to induce a primary immune response using DC from human MHC-transgenic NOD-scid mice were unsuccessful, as were direct immunization protocols. The results section, however, does highlight deficiencies that could be approached experimentally in future studies. In summary, the results presented in this thesis advance our understanding of the fundamental mechanisms controlling human cell engraftment in scid mice. This information supports the long-term goal of establishing a functional human immune system in a small animal model. We have identified many of the cell interactions and factors that regulate human cell engraftment and function in scid mice, and we have provided insights into host characteristics that will provide optimized engraftment of naïve human T cells. The studies led to the novel observations of the regulation of human CD4+ T cells by human CD8+ T cells, B cell activation, and progression of latent EBV infection to lymphoproliferative disorders in vivo. These studies further provide new information regarding trans-costimulation , a previously unrecognized mechanism of T cell activation. These results provide data on the fundamental mechanisms that underlie obstacles to the goal of achieving engraftment of a functional human immune system in scid mice

Regulation of human cell engraftment and development of EBV-related lymphoproliferative disorders in Hu-PBL-scid mice

Human PBMC engraft in mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation (Hu-PBL-scid mice). Hu-PBL-NOD-scid mice generate 5- to 10-fold higher levels of human cells than do Hu-PBL-C.B-17-scid mice, and Hu-PBL-NOD-scid beta2-microglobulin-null (NOD-scid-B2mnull) mice support even higher levels of engraftment, particularly CD4+ T cells. The basis for increased engraftment of human PBMC and the functional capabilities of these cells in NOD-scid and NOD-scid-B2mnull mice are unknown. We now report that human cell proliferation in NOD-scid mice increased after in vivo depletion of NK cells. Human cell engraftment depended on CD4+ cells and required CD40-CD154 interaction, but engrafted CD4+ cells rapidly became nonresponsive to anti-CD3 Ab stimulation. Depletion of human CD8+ cells led to increased human CD4+ and CD20+ cell engraftment and increased levels of human Ig. We further document that Hu-PBL-NOD-scid mice are resistant to development of human EBV-related lymphoproliferative disorders. These disorders, however, develop rapidly following depletion of human CD8+ cells and are prevented by re-engraftment of CD8+ T cells. These data demonstrate that 1) murine NK cells regulate human cell engraftment in scid recipients; 2) human CD4+ cells are required for human CD8+ cell engraftment; and 3) once engrafted, human CD8+ cells regulate human CD4+ and CD20+ cell expansion, Ig levels, and outgrowth of EBV-related lymphoproliferative disorders. We propose that the Hu-PBL-NOD-scid model is suitable for the in vivo analysis of immunoregulatory interactions between human CD4+ and CD8+ cells

Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice

Genetic crosses produced NOD/LtSz mice doubly homozygous for the severe combined immunodeficiency (scid) mutation and the beta2m (B2m) null allele. Both NOD/LtSz-scid/scid and NOD/LtSz-scid/scid B2m(null) mice lacked mature lymphocytes and serum Ig. However, homozygosity for the B2m(null) allele also resulted in the absence of MHC class I expression, loss of NK cell activity, accumulation of iron in the liver, and rapid clearance of human IgG1. NOD/LtSz-scid/scid B2m(null) mice supported markedly elevated levels of human T cell engraftment, compared with NOD/LtSz-scid/scid control animals, following injection with human PBMC. The increased engraftment was associated with a major increase in the number of human CD4+ T cells. Following injection with 20 million human PBMC, levels of human CD4+ T cells in the peripheral blood and spleen of NOD/ LtSz-scid/scid B2m(null) mice were 6- to 7-fold higher than those in NOD/LtSz-scid/scid mice and \u3e50-fold higher than those in C.B-17-scid/scid mice. The resulting normalization of CD4+/CD8+ ratios in NOD/LtSz-scid/scid B2m(null) mice is in sharp contrast to that observed in NOD/LtSz-scid/scid mice or in C.B-17-scid/scid mice. Circulating human IgG was cleared 6-fold more rapidly in NOD/LtSz-scid/scid B2m(null) mice than in NOD/LtSz-scid/scid mice. This rapid IgG clearance suggested a failure of the engrafted human lymphoid cells to maintain high circulating levels of human IgG. The higher levels of human CD4+ T cells and the normalization of the CD4:CD8 ratio that are observed in human PBMC-engrafted NOD/LtSz-scid/scid B2m(null) mice suggest that this system may be an excellent model for studies of HIV pathogenesis

NOD/LtSz-Rag1null mice: an immunodeficient and radioresistant model for engraftment of human hematolymphoid cells, HIV infection, and adoptive transfer of NOD mouse diabetogenic T cells

Development of a small animal model for the in vivo study of human immunity and infectious disease remains an important goal, particularly for investigations of HIV vaccine development. NOD/Lt mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation readily support engraftment with high levels of human hematolymphoid cells. However, NOD/LtSz-scid mice are highly radiosensitive, have short life spans, and a small number develop functional lymphocytes with age. To overcome these limitations, we have backcrossed the null allele of the recombination-activating gene (Rag1) for 10 generations onto the NOD/LtSz strain background. Mice deficient in RAG1 activity are unable to initiate V(D)J recombination in Ig and TCR genes and lack functional T and B lymphocytes. NOD/LtSz-Rag1null mice have an increased mean life span compared with NOD/LtSz-scid mice due to a later onset of lymphoma development, are radioresistant, and lack serum Ig throughout life. NOD/LtSz-Rag1null mice were devoid of mature T or B cells. Cytotoxic assays demonstrated low NK cell activity. NOD/LtSz-Rag1null mice supported high levels of engraftment with human lymphoid cells and human hemopoietic stem cells. The engrafted human T cells were readily infected with HIV. Finally, NOD/LtSz-Rag1null recipients of adoptively transferred spleen cells from diabetic NOD/Lt+/+ mice rapidly developed diabetes. These data demonstrate the advantages of NOD/LtSz-Rag1null mice as a radiation and lymphoma-resistant model for long-term analyses of engrafted human hematolymphoid cells or diabetogenic NOD lymphoid cells

Blockade of CD40-CD154 interferes with human T cell engraftment in scid mice

Antibodies to the ligand for CD40 (CD154) have been shown to exert profound effects on the development of cell-mediated immune responses in mice. The present study shows that an antibody to human CD154 (hCD40L) inhibits in vivo Tetanus toxoid (TT) specific secondary antibody responses in hu-PBL-scid mice, as well as the expansion of xenoreactive human T cells in the scid mice. A possible cause for the reduced expansion of xenoreactive, human T cells, was the decreased expression of murine B7.1 and B7.2 caused by the administration of anti-hCD40L. Therefore, it may be that defective maturation of murine antigen-presenting cells impeded the priming and expansion of human xenoreactive T cells