Induction of long-lived humoral immunity to poxvirus infections

Smallpox eradication through the use of a live-virus vaccine is one of the most successful public health endeavors of modern medicine. Humoral immunity against the smallpox virus, characterized by neutralizing antiviral antibody response, is stable, lasts for decades, and is considered a valuable benchmark of the functional attributes of a good vaccine. Indeed, neutralizing antibody is the best correlate of long-term protective immunity for all the currently licensed clinically effective viral vaccines. In this thesis, we investigated the contribution of host and viral factors that are responsible for the induction of long-lived antiviral humoral immunity. Crosstalk between T follicular helper (TFH) cells, T follicular regulatory (TFR) cells and B cells is essential for induction of optimal germinal center (GC) responses and subsequent high affinity antibody generation. We hypothesized that the escalating concentrations of replicating virus (antigens) provide important cues to these immune cell subsets in the GC response, and these are critical for induction and maintenance of robust, long-lived humoral immunity. To test this, we used mousepox, the disease caused by ectromelia virus (ECTV) in mice and an excellent surrogate small animal model for smallpox and generalized viral infections. We found that replication-competent wild type ECTV induced more robust TFH and GC responses, contemporaneous with significantly higher neutralizing antibody titers, compared with a poorly replicating ECTV strain. We were able to demonstrate via passive transfer-challenge experiments, that antibodies generated following infection with the poorly replicating strain were not as effective in controlling a secondary challenge as compared to antibodies generated following infection with a replicationcompetent virus. Similar differences were observed between replicating and non-replicating strains of both Vaccinia virus (VACV), vaccine-strain Modified Vaccinia Ankara (MVA) and influenza A virus. Additionally, strategies to enhance the immunogenicity of the replicating-inefficient virus by changes in antigen availability, via alterations in the dose and route of administration were evaluated. Importantly, we have discussed the ability to prospectively predict the magnitude and potency of longlived neutralizing antibody induction using immunological biomarkers in blood, which would particularly aid the field of vaccine biology. Using replication-competent and replication-poor recombinant ECTV expressing mutant versions of hen egg lysozyme (HEL) in combination with SWHEL transgenic B cells, we extended our studies to antigen-specific B cell responses in poxvirus infection. To date, affinity maturation has been studied extensively using protein or protein-hapten immunization and the current study is the first, to our knowledge, to systematically examine antigen-specific B cell responses and affinity maturation to a virus-expressed antigen during infection of a natural host under physiological conditions. In mice transferred with SWHEL B cells and infected with recombinant ECTV encoding HEL, replicating virus induced massive proliferation of SWHEL B cells, somatic hypermutations (SHM) in the variable region exon of SWHEL Ig heavy chain, and affinity maturation. In contrast, poorly replicating viruses induced minimal proliferation of SWHEL B cells with little or no SHM events. Significantly, robust SWHEL B cell responses, associated with augmented proliferation, induction of SHM events and secretion of antibody, occurred when the dose and route of administration of poorly replicating ECTV encoding HEL was altered. Collectively, the data presented in this thesis provide valuable insights into the rational design of poxviral vaccine vectors to achieve an efficacious induction of long-lasting immune memory

The orchestrated functions of innate leukocytes and t cell subsets contribute to humoral immunity, virus control, and recovery from secondary poxvirus challenge

A pivotal role for antigen-specific recall responses to secondary virus infection is well established, but the contribution of innate immune cells to this process is unknown. Recovery of mice from a primary orthopoxvirus (ectromelia virus [ECTV]) infection requires the function of natural killer (NK) cells, granulocytes, plasmacytoid dendritic cells (pDC), T cells, and B cells. However, during a secondary challenge, resolution of infection is thought to be dependent on antibody but not T cell function. We investigated the contribution of NK cells, granulocytes, and pDC to virus control during a secondary virus challenge in mice that had been primed with an avirulent, mutant strain of ECTV. Mice depleted of NK cells, granulocytes, or pDC effectively controlled virus, as did mice depleted of both CD4 and CD8 T cell subsets. However, mice concurrently depleted of all three innate cell subsets had elevated virus load, but this was significantly exacerbated in mice also depleted of CD4 and/or CD8 T cells. Increased viral replication in mice lacking innate cells plus CD4 T cells was associated with a significant reduction in neutralizing antibody. Importantly, in addition to T-dependent neutralizing antibody responses, the function of CD8 T cells was also clearly important for virus control. The data indicate that in the absence of innate cell subsets, a critical role for both CD4 and CD8 T cells becomes apparent and, conversely, in the absence of T cell subsets, innate immune cells help contain infectio

Control of chicken CR1 retrotransposons is independent of Dicer-mediated RNA interference pathway

BACKGROUND: Dicer is an RNase III-ribonuclease that initiates the formation of small interfering RNAs as a defence against genomic parasites such as retrotransposons. Despite intensive characterization in mammalian species, the biological functions of Dicer in controlling retrotransposable elements of the non-mammalian vertebrate are poorly understood. In this report, we examine the role of chicken Dicer in controlling the activity of chicken CR1 retrotransposable elements in a chicken-human hybrid DT40 cell line employing a conditional lossof- Dicer function. RESULTS: Retrotransposition is detrimental to host genome stability and thus eukaryotic cells have developed mechanisms to limit the expansion of retrotransposons by Dicer-mediated RNAi silencing pathways. However, the mechanisms that control the activity and copy numbers of transposable elements in chicken remain unclear. Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing. In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons. CONCLUSION: Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naive Mice

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status

Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo

Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.This work was funded by the National Health and Medical Research Council through the grants APP100790 (G.K) and GNT0632785 (T.N)

A Natural Genetic Variant of Granzyme B Confers Lethality to a Common Viral Infection

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining ‘Asp-ase ’ activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV

Flow cytometric characterization of neural precursor cells and their progeny

Optical fibers sensors for civil engineering are not a new idea. Their interest is based mainly on the intrinsic properties of optical fibers: electromagnetic neutrality, important capacity of multiplexing and access to long distances of measure. These sensors may cover numerous functions of the traditional sensors: detection, localization and surveillance. Thanks to interactions between the light and the optical fiber, such Brillouin scattering, the optical fiber can be on all its length, a continuously distributed sensor.The phenomenon of Brillouin scattering is well studied due to its big efficiency of scattering, its dependence towards temperature and strain and its pluri-kilometric reach. However, the double sensibility of the Brillouin frequency in temperature and strain is problematic for the simultaneous measurement of these two parameters. We shall present a possibility of discrimination of temperature and strain corresponding to the precisions wished for surveillance health monitoring.L’utilisation de capteurs à fibres optiques pour le génie civil n’est pas une idée nouvelle. Leur intérêt repose principalement sur les propriétés intrinsèques des fibres optiques: neutralité électromagnétique, capacité de multiplexage importante et accès à de longues distances de mesure. Ces capteurs sont susceptibles de couvrir de nombreuses fonctions des capteurs traditionnels : détection, localisation et surveillance. Grâce à des interactions entre la lumière et la fibre optique, telle que la diffusion Brillouin, la fibre optique peut constituer, sur toute sa longueur, un capteur continûment distribué. Le phénomène de diffusion Brillouin est très étudié de part sa grande efficacité de diffusion, sa dépendance vis-à-vis de la température et de la déformation et sa portée pluri-kilométrique. Cependant, la sensibilité de la fréquence de décalage Brillouin à la fois à la température et à la déformation rend problématique la mesure simultanée de ces deux paramètres. Nous présenterons une possibilité de discrimination de la température et de la déformation correspondant aux précisions souhaitées en terme de surveillance d'ouvrage de génie civil

Vaccine-induced protection against orthopoxvirus infection Is mediated through the combined functions of CD4 T cell-dependent antibody and CD8 T cell responses

Antibody production by B cells in the absence of CD4 T cell help has been shown to be necessary and sufficient for protection against secondary orthopoxvirus (OPV) infections. This conclusion is based on short-term depletion of leukocyte subsets in vaccinated animals, in addition to passive transfer of immune serum to naive hosts that are subsequently protected from lethal orthopoxvirus infection. Here, we show that CD4 T cell help is necessary for neutralizing antibody production and virus control during a secondary ectromelia virus (ECTV) infection. A crucial role for CD4 T cells was revealed when depletion of this subset was extended beyond the acute phase of infection. Sustained depletion of CD4 T cells over several weeks in vaccinated animals during a secondary infection resulted in gradual diminution of B cell responses, including neutralizing antibody, contemporaneous with a corresponding increase in the viral load. Long-term elimination of CD8 T cells alone delayed virus clearance, but prolonged depletion of both CD4 and CD8 T cells resulted in death associated with uncontrolled virus replication. In the absence of CD4 T cells, perforin- and granzyme A- and B-dependent effector functions of CD8 T cells became critical. Our data therefore show that both CD4 T cell help for antibody production and CD8 T cell effector function are critical for protection against secondary OPV infection. These results are consistent with the notion that the effectiveness of the smallpox vaccine is related to its capacity to induce both B and T cell memory. IMPORTANCE Smallpox eradication through vaccination is one of the most successful public health endeavors of modern medicine. The use of various orthopoxvirus (OPV) models to elucidate correlates of vaccine-induced protective immunity showed that antibody is critical for protection against secondary infection, whereas the role of T cells is unclear. Short-term leukocyte subset depletion in vaccinated animals or transfer of immune serum to naive, immunocompetent hosts indicates that antibody alone is necessary and sufficient for protection. We show here that long-term depletion of CD4 T cells over several weeks in vaccinated animals during secondary OPV challenge reveals an important role for CD4 T cell-dependent antibody responses in effective virus control. Prolonged elimination of CD8 T cells alone delayed virus clearance, but depletion of both T cell subsets resulted in death associated with uncontrolled virus replication. Thus, vaccinated individuals who subsequently acquire T cell deficiencies may not be protected against secondary OPV infection.This work was supported by National Health and Medical Research Council of Australia research grants 471426 and APP1007980 to G.K. and G.C