Yellow fever 17D as a vaccine vector for microbial CTL epitopes: protection in a rodent malaria model

The yellow fever vaccine 17D (17D) is safe, and after a single immunizing dose, elicits long-lasting, perhaps lifelong protective immunity. One of the major challenges facing delivery of human vaccines in underdeveloped countries is the need for multiple injections to achieve full efficacy. To examine 17D as a vector for microbial T cell epitopes, we inserted the H-2Kd–restricted CTL epitope of the circumsporozoite protein (CS) of Plasmodium yoelii between 17D nonstructural proteins NS2B and NS3. The recombinant virus, 17D-Py, was replication competent and stable in vitro and in vivo. A single subcutaneous injection of 105 PFU diminished the parasite burden in the liver by ∼70%. The high level of protection lasted between 4 and 8 wk after immunization, but a significant effect was documented even 24 wk afterwards. Thus, the immunogenicity of a foreign T cell epitope inserted into 17D mimics some of the remarkable properties of the human vaccine. Priming with 17D-Py followed by boosting with irradiated sporozoites conferred sterile immunity to 90% of the mice. This finding indicates that the immune response of vaccine-primed individuals living in endemic areas could be sustained and magnified by the bite of infected mosquitoes

Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses

Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses

Efficient development of plasmodium liver stage-specific memory CD8+ T cells during the course of blood-stage malarial infection.

Immunity to Plasmodium liver stages in individuals in malaria-endemic areas is inextricably linked to concomitant blood-stage parasitemia. Although Plasmodium sporozoite infection induces measurable CD8+ T cell responses, the development of memory T cells during active erythrocytic infection remains uncharacterized. Using transgenic T cells, we assessed antigen-specific effector CD8+ T cell responses induced by normal (NorSpz) and radiation-attenuated (IrrSpz) Plasmodium yoelii sporozoites. The magnitude, phenotypic activation, and differentiation pathway of CD8+ T cells were similarly induced by NorSpz and IrrSpz. Moreover, in normal mice, memory T cells elicited after priming with NorSpz and IrrSpz generated identical recall responses after a heterologous boost strategy. Furthermore, these recall responses exhibited comparable in vivo antiparasite activity. Our results indicate that sporozoites that retain their infective capacity induce memory CD8+ T cells that are robustly recalled by secondary immunization. Thus, erythrocytic infection does not preclude the establishment of memory CD8+ T cell responses to malarial liver stages

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle

Monoclonal Antibodies against Plasmodium falciparum Circumsporozoite Protein

Malaria is a mosquito-borne infectious disease caused by the parasite Plasmodium spp. Malaria continues to have a devastating impact on human health. Sporozoites are the infective forms of the parasite inside mosquito salivary glands. Circumsporozoite protein (CSP) is a major and immunodominant protective antigen on the surface of Plasmodium sporozoites. Here, we report a generation of specific monoclonal antibodies that recognize the central repeat and C-terminal regions of P. falciparum CSP. The monoclonal antibodies 3C1, 3C2, and 3D3—specific for the central repeat region—have higher titers and protective efficacies against challenge with sporozoites compared with 2A10, a gold standard monoclonal antibody that was generated in early 1980s

Polarity of Chromosome XII and the Nucleolus.

<p>(A) Distribution and coherence of the nucleolus. The top panels illustrate the relative behavior of a nucleolar protein (Gar1-GFP) and Htb2-mRFP during entry into anaphase of a normal cell cycle. Note that a limited amount of chromatin emerged into the bud domain at an early time-point (*) after being constricted at the level of the bud neck. The nucleolar signal then follows slowly, remaining in contact with the stretched chromatin mass. It subsequently is resolved into two equal parts that associate with the chromatin masses. Strain: ATY2416. The lower panels illustrate an intermediate time point when two nucleolar markers can be seen to traverse the neck during the normal cell cycle. These are the condensin, (Brn1-GFP) (that coats compacted rDNA and defines a narrow thread), and Sik1-mRFP, that occupies a broader territory. Both markers are compressed as the nucleolus traverses the bud neck region. Strain: ATY7473. (B) Schematic representation of nuclear domains and chromosome XII in arrested cells. The diagram illustrates the anticipated position of chromosome XII when the nucleolus (green) abuts on the bud neck. The position of the septin ring is also indicated, suggesting that this ring—in conjunction with the nucleolus—could control passage across the neck. The lacO or tetO loci that we have followed are from the following strains: locus A (ATY7633), locus B (ATY7634), locus C (ATY7733), locus D (ATY7241), locus E (ATY7637) and locus F (ATY7102). (C) Distribution and organization of telomere XII after arrest. Images of a <i>MET3-CDC20</i> strain that expresses Htb2-mRFP, Net1-CFP and a tetO tag at locus F (TelR). In the arrested cells, the tail portion of the chromatin and Net1-CFP (blue) were in the mother domain, whereas the mass of chromatin was in the bud domain. Note that when the tail was fully extended, the red extremity and the tetO locus were distal to Net1-CFP. The portion of the Htb2-mRFP signal corresponding to the nucleolus was relatively faint, perhaps because it is actively transcribed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174306#pone.0174306.ref036" target="_blank">36</a>]. Strain: ATY7102. (D) Distribution of rDNA: Images of a <i>MET3-CDC20</i> strain that expresses Htb2-mRFP, Net1-CFP and a tetO tag at locus D. Cells were arrested by addition of methionine for 4 hours and then imaged. Note that, by contrast to cells with a tagged telomere, the tagged locus abutted on the Net1-CFP signal and that—when the tail was maximally extended—this locus was not within the red telomeric region. Strain: ATY7241. (E) Distribution and organization of the telomeres of chromosome V: A <i>MET3-CDC20</i> strain expressing Htb2-mRFP, TelVL-tetO and TelVR-lacO along with YFP-tetR and CFP-lacI was arrested by addition of methionine for 4 hours. In some cells, both TelVL-YFP and TelVR-CFP were located in a single domain (left), while in other cells, the signals were each in a different domain (right). Strain: ATY7272. (F) Concanavalin staining of cells: Cells were stained with rhodamine-conjugated ConA for 15 minutes on ice. The cells were then washed and reincubated. The diagram indicates the staining pattern that was observed before and after reincubation to allow bud growth. (G) Localization of Gar1-GFP in <i>rpa190-3</i> ts mutant cells: <u>Images on the left</u>: A <i>MET3-CDC20 rpa190-3</i> strain expressing Gar1-GFP was labeled with rho-Con A and arrested by addition of methionine at 23°C. The cells were then shifted to 36°C (or not) and studied after 0–4 hours. The two upper images show that, before reincubation, Gar1-GFP remained in the mother domain (encircled by red) and reached as far as the bud neck. The three lower images are examples in which Gar1-GFP was also detected in the bud domain after a 4 hour reincubation. The bar graphs on the right quantitate the distribution of Gar1-GFP in the same ts cells and also in isogenic Ts+ cells. Note the stability of arrest in the Ts+ cells at both temperatures and the progressive loss of arrest in the ts cells after incubation at 36°C. The distribution of Gar1-GFP is symbolized as follows: M: only in the mother domain, N: contacts the neck or spans the neck, B: only in the bud, M > B: in both domains with more signal in the mother domain, M ~ B: approximately equal in both domains. In this figure and all others that quantitate distribution of markers, the data are given ± one standard deviation. Statistical significance was evaluated by Student’s <i>t</i>-test. In the lower right panel, pairs of bars (T0 hr, T4 hr) bearing the same number of stars have been compared to each other. *P < 0.005, **P < 0.05, and ***P < 0.05. Strains: ATY7760 and ATY3175. Parallel studies of <i>rpa190-3</i> cells that express a tagged histone (Htb2-GFP) did not show any indication of progression to or beyond anaphase.</p

Dynamics of Chromatin and the Nucleolus in Arrested Cells.

<p>(A) Time-lapse images of chromatin and the nucleolus. A <i>MET3-CDC20</i> strain expressing the tagged histone, Htb2-mRFP, and the tagged nucleolar protein, Gar1-GFP, was arrested by addition of methionine for 4 hours before imaging. Note that the chromatin mass (red) shifted between the mother (M) and bud (B) domains repeatedly and changed shape in doing so. When most of the chromatin was in the bud domain, a narrow tail (T) extended across the mother domain. When the chromatin was retracting from the bud domain, the trailing edge often had a flattened surface (F) (rectangle). The nucleolus (Nu—green) always remained in the mother domain. In the inserts, the green signal is absent so that the red can be more uniformly detected. The blue images are bright-field. Strain: ATY3175. (B) As in Fig 2A except that the nucleolus and chromatin in the mother domain were further extended. In this situation, the tail in the mother domain defined a narrow ribbon that ends in a red patch (*) at its extremity. Strain: ATY3175. (C) Time-lapse images of chromatin, the spindle and the spindle pole body. A <i>MET3-CDC20</i> strain expressing Htb2-mRFP, Tub1-GFP and the SPB protein, Spc42-CFP, was arrested by addition of methionine for 4 hours and then imaged. The orientation of the spindle (Sp) shifted and the entire spindle frequently moved from one domain to the other along with chromatin. As shown in the lower images, the SPBs (arrow) remained associated with both extremities of the spindle. Strain: ATY7135. (D-E) Localization of centromeres (D) and kinetochores (E). The tagged variant histone, Cse4-GFP, and the tagged kinetochore protein, Ndc10-GFP, were localized in cycling cells (upper panels) and in a <i>MET3-CDC20</i> strain after arrest for 4 hours by addition of methionine (lower panels). Cycling cells: In most unbudded cells (Unb), both Cse4-GFP and Ndc10-GFP formed single clusters, while they had doubled in cells with small buds (Sm) or medium-sized buds (Md) and in cells in anaphase (Ana). Arrested cells: Note the frequent presence of additional fluorescent foci (fragments) for both Cse4-GFP and Ndc10-GFP. Both cells expressing Cse4-GFP with the tag at an internal position and cells with the tag at the C-terminus exhibited comparable fragmentation. Strains: ATY6196, ATY6882, ATY6833. (F) Quantitation of the number of foci detected in preparations equivalent to Fig 2D and 2E. Strains: ATY6196 and ATY6882.</p

Organization of the Nuclear Envelope in Arrested Cells.

<p>(A) Dynamics of the nuclear envelope: Time-lapse images of a <i>MET3-CDC20</i> strain expressing Htb2-mRFP and Nup49-GFP, after addition of methionine for 4 hours to deplete Cdc20. Note that the NE extends approximately equally into the mother and bud at all time points. The star (*) indicates a region of the nucleoplasm that is occupied by the nucleolus, judging from examination of other arrested strains. M and B designate the mother and bud domains respectively. In these and all other images, the scale bar is 5 microns. Unless indicated to the contrary, all images are projections of 5–10 Z-sections. In this figure and all others, the bud neck is designated as BN. Strain: ATY4435. (B) The bud neck: Distribution of Nup49-GFP. A <i>MET3-CDC20</i> strain expressing Htb2-mRFP and Nup49-GFP was arrested for 4 hr. The arrows indicate the frequent absence of Nup49-GFP from the bud neck. Strain: ATY4435. Exclusion of DiOC6 from the bud neck. A petite <i>MET3-CDC20</i> strain expressing Htb2-mRFP was arrested and stained with DiOC6 to detect lipids of the ER (and possibly other membranes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174306#pone.0174306.ref017" target="_blank">17</a>]). Note the near-absence of signal at the bud neck. This image is from a single 0.5 micron Z-section. Strain: ATY4435.</p

A New Method to Determine Antigen-Specific CD8+ T Cell Activity in Vivo by Hydrodynamic Injection

Hydrodynamic tail vein (HTV) delivery is a simple and rapid tail vein injection method of a high volume of naked plasmid DNA resulting in high levels of foreign gene expression in organs, especially the liver. Compared to other organs, HTV delivery results in more than a 1000-fold higher transgene expression in liver. After being bitten by malaria-infected mosquitoes, malaria parasites transiently infect the host liver and form the liver stages. The liver stages are known to be the key target for CD8+ T cells that mediate protective anti-malaria immunity in an animal model. Therefore, in this study, we utilized the HTV delivery technique as a tool to determine the in vivo cytotoxic effect of malaria antigen-specific CD8+ T cells. Two weeks after mice were immunized with recombinant adenoviruses expressing malarial antigens, the immunized mice as well as naïve mice were challenged by HTV delivery of naked plasmid DNA co-encoding respective antigen together with luciferase using dual promoters. Three days after the HTV challenge, non-invasive whole-body bioluminescent imaging was performed. The images demonstrate in vivo activity of CD8+ T cells against malaria antigen-expressing cells in liver