Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

Role of T cell response during vaccine immunity to tuberculosis

Tuberculosis remains one of the leading causes of death in man due to a single infectious agent. An estimated one-third of the world's population is infected with the causative agent, Mycobacterium tuberculosis (Mtb), despite the availability of the widely used vaccine, BCG. BCG has significantly varying protection rates with the lowest level of protection seen with the most common form of TB, adult pulmonary TB. Thus, numerous studies are being conducted to develop a more efficient vaccine. The ideal candidate vaccine would possess the ability to induce a solid and strong Th1 response, as this is the subset of T cells primarily involved in clearance of the infection. A novel vaccine should also induce such a response that may be recalled and expanded upon subsequent infection. Our group has introduced a mutant of a virulent strain of Mtb which lacks a component of the immunogenic antigen 85 complex (Ag85). Our vaccine, ΔfbpA, does not secrete the fibronectin binding protein Ag85A, and this has shown to lead to its attenuation in both murine macrophages and mice. Previous studies have also proven that ΔfbpA is more protective in mice than BCG against virulent aerosol challenge with Mtb. This study addresses the mechanisms of protection observed with ΔfbpA by phenotyping responding T cells. We first evaluated the ability of dendritic cells to present the mycobacteria to naïve T cells, an in vitro mock of primary immunization. We also measured the response of primed T cells to macrophage-presented mycobacteria to interpret the possible response of a vaccinated host to a boost. We concluded that ΔfbpA can elicit a stronger Th1 response compared to BCG in vitro, and further observed that this enhanced response is at least partly due to the presence of proteins encoded by a region of the genome absent in all strains of BCG. Finally, we observed this heightened Th1 response in the mouse model after primary vaccination and a virulent aerosol challenge. The cytolytic T cell response was also measured after virulent challenge and was found to be superior in the ΔfbpA-treated group when compared to the BCG group. ^

MODULATION OF HOST INTESTINAL MYOELECTRIC ACTIVITY BY LOCAL ANAPHYLAXIS: A COMPONENT OF PROTECTIVE IMMUNITY TO AN ENTERIC PARASITE (TRICHINELLA SPIRALIS, EIMERIA NIESCHULZI, BOWEL MOTILITY)

The hypothesis tested was that rapid rejection of Trichinella spiralis infective larvae from immunized rats following a challenge infection is associated with a local anaphylactic reaction, and this response should be reflected in altered small intestinal motility. The objective was to determine if altered gut smooth muscle function accompanies worm rejection based on the assumption that anaphylaxis in vivo could be detected by changes in intestinal smooth muscle contractile activity (ie. an equivalent of the Schultz-Dale reaction or in vitro anaphylaxis). The aims were to (1) characterize motility changes by monitoring intestinal myoelectric activity in conscious rats during the enteric phase of T. spiralis infection in immunized hosts, (2) detect the onset and magnitude of myoelectric changes caused by challenge infection in immunized rats, (3) determine the parasite stimulus causing changes, and (4) determine the specificity of host response to stimulation. Electrical slow wave frequency, spiking activity, normal interdigestive migrating myoelectric complexes and abnormal migrating action potential complexes were measured. Changes in myoelectric parameters induced by larvae inoculated into the duodenum of immune hosts differed from those associated with primary infection with respect to time of onset, magnitude and duration. Myoelectric changes elicited by live larvae could not be reproduced by inoculation of hosts with dead larvae, larval excretory-secretory products, or by challenge with a heterologous parasite, Eimeria nieschulzi. These results indicate that (1) local anaphylaxis is a component of the initial response to T. spiralis in immune hosts, since the rapid onset of altered smooth muscle function parallels in time the expression of rapid rejection of infective larvae, and (2) an active mucosal penetration attempt by the worm is necessary to elicit this host response. These findings provide evidence that worm rejection is a consequence of, or sequel to, an immediate hypersensitivity reaction elicited when parasites attempt to invade the gut mucosa of immunized hosts. ^

Effects of breastfeeding and naturally acquired immunity on infection with {\it Giardia lamblia}: A study in a cohort of Mexican infants

Giardia lamblia is one of the most common causes of gastrointestinal tract infection among young children worldwide. Yet host protection against this parasite and the effect of infection with Giardia on infant growth are poorly understood. It was hypothesized that among young children, protection against infection with Giardia is afforded by breastfeeding and previous infection with the parasite and further, that infection with Giardia decreases growth velocity. From 4/88 to 4/90, 197 infants in a poor area of Mexico City were followed from 0 to 18 months of age, with stool specimens, symptoms and feeding status data collected weekly. A total of 6,031 stool specimens were tested for Giardia antigen by enzyme-linked immunosorbent assay. There were 1.0 Giardia infections per child-year; 25% were symptomatic and 54% lasted more than 1 month; 94 infants had 1, and 33 had 2 or more infections. Breastfeeding status was coded and analyzed for each child-week of follow up. 91% of study infants were breastfed from birth, 57% at 6 months and 38% at 12 months of age. Rate ratios for non-breastfeeding adjusted for confounding factors were calculated from stratified analyses and the Cox proportional hazards model. Not breastfeeding was a significant risk factor for first infection with Giardia vs. any breastfeeding (adjusted RR = 1.8; 1.1, 2.8) at all ages; a dose response was demonstrated by degree of breastfeeding. The adjusted rate ratio for non-breastfeeding vs. partial breastfeeding was 1.6 (1.03, 2.6) and for non-breastfeeding vs. complete breastfeeding was 4.7 (1.4, 15.9). Among Giardia infected infants, breastfeeding did not protect against diarrheal symptoms or shorten the duration of carriage. First and repeat infections with Giardia did not differ in duration or the percent symptomatic. The analysis of growth and Giardia infection was inconclusive but suggested that a history of Giardia infection might be associated with decreased weight velocity, while an immediate chronic infection might be associated with increased weight velocity. In summary, these data indicate that breastfeeding protects infants against infection with Giardia; provide no evidence of protection against repeat infections resulting from a prior infection and suggest but do not establish that a history of Giardia infection might be associated with decreased growth in young children. ^

INDUCTION OF STRONGER AND LONG LASTING VACCINE IMMUNITY TO TUBERCULOSIS

Tuberculosis is a major cause of death due to an infection in mankind. BCG vaccine protects against childhood tuberculosis although, it fails to protect against adult tuberculosis. BCG vaccine localizes to immature phagosomes of macrophages, and avoids lysosomal fusion, which decreases peptide antigen production. Peptides are essential for macrophage-mediated priming of CD4 and CD8 T cells respectively through MHC-II and MHC-I pathways. Furthermore, BCG reduces the expression of MHC-II in macrophages of mice after infection, through Toll-like receptor-1/2 (TLR-1/2) mediated signaling. In my first aim, I hypothesized that BCG-induced reduction of MHC-II levels in macrophages can decrease CD4 T cell function, while activation of other surface Toll-like receptors (TLR) can enhance CD4 T cell function. An in vitro antigen presentation model was used where, TLR activated macrophages presented an epitope of Ag85B, a major immunogen of BCG to CD4 T cells, and T cell derived IL-2 was quantitated as a measure of antigen presentation. Macrophages with BCG were poor presenters of Ag85B while, TLR-7/9/5/4 and 1/2 activation led to an enhanced antigen presentation. Furthermore, TLR-7/9 activation was found to down-regulate the degradation of MHC-II through ubiquitin ligase MARCH1, and also stimulate MHC-II expression through activation of AP-1 and CREB transcription elements via p38 and ERK1/2 MAP kinases. I conclude from Aim-I studies that TLR-7/9 ligands can be used as more effective ‘adjuvants’ for BCG vaccine. In Aim-II, I evaluated the poor CD8 T cell function in BCG vaccinated mice thought to be due to a decreased leak of antigens into cytosol from immature phagosomes, which reduces the MHC-I mediated activation of CD8 T cells. I hypothesized that rapamycin co-treatment could boost CD8 T cell function since it was known to sort BCG vaccine into lysosomes increasing peptide generation, and it also enhanced the longevity of CD8 T cells. Since CD8 T cell function is a dynamic event better measurable in vivo, mice were given BCG vaccine with or without rapamycin injections and challenged with virulent Mycobacterium tuberculosis. Organs were analysed for tetramer or surface marker stained CD8 T cells using flow cytometry, and bacterial counts of organisms for evaluation of BCG-induced protection. Co-administration of rapamycin with BCG significantly increased the numbers of CD8 T cells in mice which developed into both short living effector- SLEC type of CD8 T cells, and memory precursor effector-MPEC type of longer-living CD8 T cells. Increased levels of tetramer specific-CD8 T cells correlated with a better protection against tuberculosis in rapamycin-BCG group compared to BCG vaccinated mice. When rapamycin-BCG mice were rested and re-challenged with M.tuberculosis, MPECs underwent stronger recall expansion and protected better against re-infection than mice vaccinated with BCG alone. Since BCG induced immunity wanes with time in humans, we made two novel observations in this study that adjuvant activation of BCG vaccine and rapamycin co-treatment both lead to a stronger and longer vaccine-mediated immunity to tuberculosis.

Protective immunity to HIV-1 in SCID/beige mice reconstituted with peripheral blood lymphocytes of exposed but uninfected individuals

Immunodeficiency typically appears many years after initial HIV infection. This long, essentially asymptomatic period contributes to the transmission of HIV in human populations. In rare instances, clearance of HIV-1 infection has been observed, particularly in infants. There are also reports of individuals who have been frequently exposed to HIV-1 but remain seronegative for the virus, and it has been hypothesized that these individuals are resistant to infection by HIV-1. However, little is known about the mechanism of immune clearance or protection against HIV-1 in these high-risk individuals because it is difficult to directly demonstrate in vivo protective immunity. Although most of these high-risk individuals show an HIV-1-specific cell-mediated immune response using in vitro assays, their peripheral blood lymphocytes (PBLs) are still susceptible to HIV infection in tissue culture. To study this further in vivo, we have established a humanized SCID mouse infection model whereby T-, B-, and natural killer-cell defective SCID/beige mice that have been reconstituted with normal human PBLs can be infected with HIV-1. When the SCID/beige mice were reconstituted with PBLs from two different multiply exposed HIV-1 seronegative individuals, the mice showed resistance to infection by two strains of HIV-1 (macrophage tropic and T cell tropic), although the same PBLs were easily infected in vitro. Mice reconstituted with PBLs from non-HIV-exposed controls were readily infected. When the same reconstituted mice were depleted of human CD8 T cells, however, they became susceptible to HIV-1 infection, indicating that the in vivo protection required CD8 T cells. This provides clear experimental evidence that some multiply exposed, HIV-1-negative individuals have in vivo protective immunity that is CD8 T cell-dependent. Understanding the mechanism of such protective immunity is critical to the design and testing of effective prophylactic vaccines and immunotherapeutic regimens.

Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections

Chronic Pseudomonas aeruginosa infection occurs in 75–90% of patients with cystic fibrosis (CF). It is the foremost factor in pulmonary function decline and early mortality. A connection has been made between mutant or missing CF transmembrane conductance regulator (CFTR) in lung epithelial cell membranes and a failure in innate immunity leading to initiation of P. aeruginosa infection. Epithelial cells use CFTR as a receptor for internalization of P. aeruginosa via endocytosis and subsequent removal of bacteria from the airway. In the absence of functional CFTR, this interaction does not occur, allowing for increased bacterial loads in the lungs. Binding occurs between the outer core of the bacterial lipopolysaccharide and amino acids 108–117 in the first predicted extracellular domain of CFTR. In experimentally infected mice, inhibiting CFTR-mediated endocytosis of P. aeruginosa by inclusion in the bacterial inoculum of either free bacterial lipopolysaccharide or CFTR peptide 108–117 resulted in increased bacterial counts in the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. There was a significant decrease in translocation of this organism to the gastrointestinal submucosa in transgenic mice that are heterozygous carriers of a mutant ΔF508 CFTR allele, suggesting heterozygous CFTR carriers may have increased resistance to typhoid fever. The identification of CFTR as a receptor for bacterial pathogens could underlie the biology of CF lung disease and be the basis for the heterozygote advantage for carriers of mutant alleles of CFTR.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria

Individuals living in regions where malaria is endemic develop an acquired immunity to malaria which enables them to remain asymptomatic while still carrying parasites. Field studies indicate that cumulative exposure to a variety of diverse Plasmodium parasites is required for the transition from symptomatic to asymptomatic malaria. This study used a simulation model of the within-host dynamics of P. falciparum to investigate the development of acquired clinical immunity under different transmission conditions and levels of parasite diversity. Antibodies developed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), a clonally variant molecule, were assumed to be a key human immunological response to P. falciparum infection, along with responses to clonally conserved but polymorphic antigens. The time to the development of clinical immunity was found to be proportional to parasite diversity and inversely proportional to transmission intensity. The effect of early termination of symptomatic infections by chemotherapy was investigated and found not to inhibit the host's ability to develop acquired immunity. However, the time required to achieve this state was approximately double that compared to when no treatment was administered. This study demonstrates that an immune response primarily targeted against PfEMP1 has the ability to reduce clinical symptoms of infections irrespective of whether treatment is administered, supporting its role in the development of acquired clinical immunity. The results also illustrate a novel use for simulation models of P. falciparum infections, investigation of the influence of intervention strategies on the development of naturally acquired clinical immunity.

New insights on the development of fungal vaccines: from immunity to recent challenges

Fungal infections are emerging as a major problem in part due to high mortality associated with systemic infections, especially in the case of immunocompromised patients. With the development of new treatments for diseases such as cancer and the acquired immune deficiency syndrome pandemic, the number of immunosuppressed patients has increased and, as a consequence, also the number of invasive fungal infections has increased. Several studies have proposed new strategies for the development of effective fungal vaccines. In addition, better understanding of how the immune system works against fungal pathogens has improved the further development of these new vaccination strategies. As a result, some fungal vaccines have advanced through clinical trials. However, there are still many challenges that prevent the clinical development of fungal vaccines that can efficiently immunise subjects at risk of developing invasive fungal infections. In this review, we will discuss these new vaccination strategies and the challenges that they present. In the future with proper investments, fungal vaccines may soon become a reality.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale

Background: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus) microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. Results: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. Conclusions: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Acute susceptibility of aged mice to infection with Candida albicans

The effect of aging on host resistance to systemic candidosis was assessed by monitoring the course of infection in 16-month-old CBA/CaH mice (aged non-immune) and in a comparable group that had been infected with a sublethal dose of Candida albicans at 6 weeks of age (aged immune). Aged non-immune mice showed rapid progression of the disease, with a marked increase in the number of mycelia in the brain and kidney, and early morbidity, Foci of myocardial necrosis were evident, but inflammatory cells were sparse. The histological picture in the aged immune mice was similar to that in the aged non-immune group, although fewer mycelial aggregates were seen. Both groups of aged mice showed a significantly lower fungal burden in the brain on day 1 of infection, but on day 4, colony counts increased significantly in the aged non-immune mice, Comparison of cytokine gene expression in the infected brains showed that the relative amount of interferon-gamma and tumour necrosis factor-alpha cDNA were similar in all three groups. Interleukin-6 was elevated in both infected non-immune and uninfected aged mice. Aged immune mice showed no morbidity after challenge, and both colonisation and tissue damage were reduced in comparison with the aged non-immune animals.

Comparative Study of the Standard Fluorescent Antibody to Membrane Antigen (FAMA) Assay and a Flow Cytometry-Adapted FAMA Assay To Assess Immunity to Varicella-Zoster Virus

A flow cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster virus (VZV) was developed and tested in 62 serum samples, showing 90.32% accuracy obtained from a receiver operating characteristic (ROC) curve with a 0.9125 (95% confidence interval [CI], 0.829 to 1.00) area below the curve compared to the result with standard FAMA.

Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Immunity to asexual blood stage malaria and vaccine approaches

The development of a malaria vaccine seems to be a definite possibility despite the fact that even individuals with a life time of endemic exposure do not develop sterile immunity. An effective malaria vaccine would be invaluable in preventing malaria-associated deaths in endemic areas, especially amongst children less than 5 years of age and pregnant women. This review discusses our current understanding of immunity against the asexual blood stage of malaria - the stage that is responsible for the symptoms of the disease - and approaches to the design of an asexual blood stage vaccine.