Juan Rodríguez

Quantification of antigen specific CD8+ T cells using an ELISPOT assay

An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.

Cellular targets and mechanisms of nitros(yl)ation: An insight into their nature and kinetics in vivo

There is mounting evidence that the established paradigm of nitric oxide (NO) biochemistry, from formation through NO synthases, over interaction with soluble guanylyl cyclase, to eventual disposal as nitrite/nitrate, represents only part of a richer chemistry through which NO elicits biological signaling. Additional pathways have been suggested that include interaction of NO-derived metabolites with thiols and metals to form S-nitrosothiols (RSNOs) and metal nitrosyls. Despite the overwhelming attention paid in this regard to RSNOs, little is known about the stability of these species, their significance outside the circulation, and whether other nitros(yl)ation products are of equal importance. We here show that N-nitrosation and heme-nitrosylation are indeed as ubiquitous as S-nitrosation in vivo and that the products of these reactions are constitutively present throughout the organ system. Our study further reveals that all NO-derived products are highly dynamic, have fairly short lifetimes, and are linked to tissue oxygenation and redox state. Experimental evidence further suggests that nitroso formation occurs substantially by means of oxidative nitrosylation rather than NO autoxidation, explaining why S-nitrosation can compete effectively with nitrosylation. Moreover, tissue nitrite can serve as a significant extravascular pool of NO during brief periods of hypoxia, and tissue nitrate/nitrite ratios can serve as indicators of the balance between local oxidative and nitrosative stress. These findings vastly expand our understanding of the fate of NO in vivo and provide a framework for further exploration of the significance of nitrosative events in redox sensing and signaling. The findings also raise the intriguing possibility that N-nitrosation is directly involved in the modulation of protein function.

A heterologous prime-boost regime using DNA and recombinant vaccinia virus expressing the Leishmania infantum P36/LACK antigen protects BALB/c mice from cutaneous leishmaniasis.

A heterologous prime-boost vaccination with DNA vectors and vaccinia virus recombinants (VVr) has been shown to enhance specific cellular immune responses and to elicit significant protection against pathogens in animal models. In this study, we have analyzed, in the leishmaniasis cutaneous murine model, the effectiveness of this prime-boost strategy by immunizing with a DNA vector followed by boost with a VVr expressing the same Leishmania infantum P36/LACK antigen. After DNA priming and VVr boost, we challenged susceptible BALB/c mice with live L. major promastigotes, and examined the increase in footpad lesion size and parasite load in draining lymph nodes. Compared to controls, we observed reduction of up to 70% in lesion size and 1000-fold in parasite load. DNA prime-VVr boost before challenge elicited a Th1 type immune response in spleen cells from immunized animals. This DNA/VVr vaccination approach could be of utility in the prophylaxis against leishmaniasis.

Enhanced CD8+ T cell response to HIV-1 env by combined immunization with influenza and vaccinia virus recombinants

With the aim to determine if immunization with two different live recombinant viral vectors could lead to an enhancement of the cellular immune response to HIV-1 antigens, we have characterized the CD8+ T cell response elicited against the V3 loop epitope from HIV-1 env protein in Balb/c mice immunized with either: a recombinant influenza virus (Flu-Env) expressing the V3 loop epitope from HIV-1 strain IIIB, a vaccinia virus recombinant (VV-Env) expressing the complete HIV-1-IIIB env protein, or a combination of both. The CD8+ T cell response, measured by the ELISPOT assay, in animals primed with Flu-Env and boosted with VV-Env was 5 to 6 times higher than in animals inoculated with either Flu-Env or VV-Env alone. Similar results were obtained with recombinant viruses expressing the V3 loop epitope or the complete env protein, respectively, from the MN strain of HIV-1. Our results indicate that the use of two different live vectors for priming and boosting has a synergistic effect on the immune response against HIV-1, and could represent a novel vaccination strategy against AIDS.

Sex recognition by odour and variation in the uropygial gland secretion in starlings

1. Although a growing body of evidence supports that olfaction based on chemical compounds emitted by birds may play a role in individual recognition, the possible role of chemical cues in sexual selection of birds has been only preliminarily studied. 2. We investigated for the first time whether a passerine bird, the spotless starling Sturnus unicolor, was able to discriminate the sex of conspecifics by using olfactory cues and whether the size and secretion composition of the uropygial gland convey information on sex, age and reproductive status in this species. 3. We performed a blind choice experiment during mating, and we found that starlings were able to discriminate the sex of conspecifics by using chemical cues alone. Both male and female starlings preferred male scents. Furthermore, the analysis of the chemical composition of the uropygial gland secretion by using gas chromatography-mass spectrometry (GC-MS) revealed differences between sexes, ages and reproductive status. 4. In conclusion, our study reveals for first time that a passerine species can discriminate the sex of conspecifics by relying on chemical cues and suggests that the uropygial gland secretion may potentially function as a chemical signal used in mate choice and/or intrasexual competition in this species.

Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL- 12 in combination with purified p36

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.

Induction of Protective Immunity against Malaria by Priming-Boosting Immunization with Recombinant Cold-Adapted Influenza and Modified Vaccinia Ankara Viruses Expressing a CD8+-T-Cell Epitope Derived from the Circumsporozoite Protein of Plasmodium yoelii

ABSTRACT We immunized mice with an attenuated (cold-adapted) influenza virus followed by an attenuated vaccinia virus (modified vaccinia virus Ankara), both expressing a CD8+-T-cell epitope derived from malaria sporozoites. This vaccination regimen elicited high levels of protection against malaria. This is the first time that the vaccine efficacy of a recombinant cold-adapted influenza virus vector expressing a foreign antigen has been evaluated.

Visual cues and parental favouritism in a nocturnal bird

Visual signals are crucial for parent–offspring communication, although their functioning has been neglected for nocturnal birds. Here, we investigated parental preference for nestling coloration in nocturnal conditions—a question hitherto unexplored—in a nocturnal raptor, the scops owl (Otus scops). We assessed how parents allocated food during the night in relation to a manipulation of ultraviolet (UV) reflectance of the cere (skin above the beak) of their offspring. Reflectance of the cere shows a marked peak in the UV part of the spectrum, and location of the UV peak is related to nestling body mass (i.e. heavier nestlings have a UV peak at lower wavelengths). We found evidence of parental bias in favour of lighter offspring: UV-reduced nestlings gained more weight during the night than their control siblings. This study provides the first experimental evidence of the use of visual cues for parent–offspring communication in a nocturnal bird.

Parental favouritism strategies in the asynchronously hatching European Roller ( Coracias garrulus )

In altricial birds, resource allocation during early developmental stages is the result of an interaction between parental feeding decisions and scramble competition between nestmates. Hatching asynchrony in birds leads to a pronounced age hierarchy among their offspring. Therefore, whenever parents exert control over resource allocation parents feeding asynchronous broods should simultaneously assess individual offspring internal condition and age. In this study, we first studied whether the highly ultraviolet (UV) reflective body skin of nestlings in the asynchronous European Roller (Coracias garrulus; roller hereafter) relates to nestling quality. In a second stage, we experimentally studied parental biases in food allocation towards senior and junior sibling rollers in relation to a manipulation of UV reflectance of the skin of their offspring. Heavier roller nestlings had less brilliant and less UV saturated skins than weaker nestlings. In our experiment, we found that parents with large broods preferentially fed nestlings presenting skin coloration revealing small body size (i.e. control nestlings) over nestlings presenting skin coloration revealing large body size (i.e. UV-blocked nestlings). Within the brood, we found that parental food allocation strategy depended on nestling age: parents preferentially fed senior nestlings signalling small body size, but did not show preference between control and UV-blocked junior nestlings. These results emphasise that parent rollers use UV cues of offspring quality while balancing the age of their offspring to adjust their feeding strategies, and suggest that parents may adopt finely tuned strategies of control over resource allocation in asynchronous broods.

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8+ T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.