Pre-clinical evaluation of a P. berghei-based whole-sporozoite malaria vaccine candidate.

Whole-sporozoite vaccination/immunization induces high levels of protective immunity in both rodent models of malaria and in humans. Recently, we generated a transgenic line of the rodent malaria parasite P. berghei (Pb) that expresses the P. falciparum (Pf) circumsporozoite protein (PfCS), and showed that this parasite line (PbVac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human Pf parasite. Here, we analyzed PbVac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of PbVac parasites was generated through a process of cyclic propagation and clonal expansion in mice and mosquitoes and was genetically characterized. A highly sensitive qRT-PCR-based method was established that enables PbVac parasite detection and quantification at low parasite densities in vivo. This method was employed in a biodistribution study in a rabbit model, revealing that the parasite is only present at the site of administration and in the liver up to 48 h post infection and is no longer detectable at any site 10 days after administration. An extensive toxicology investigation carried out in rabbits further showed the absence of PbVac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of PbVac were completely eliminated by Malarone® treatment. Collectively, our pre-clinical safety assessment demonstrates that PbVac possesses all characteristics necessary to advance into clinical evaluation.

A Plasmodium berghei sporozoite-based vaccination platform against human malaria.

There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.

Reversing gene expression in cardiovascular target organs following chronic depression of the paraventricular nucleus of hypothalamus and rostral ventrolateral medulla in spontaneous hypertensive rats.

BACKGROUND: Chronic overexpression of an inwardly rectifying potassium channel (hKir2.1) in the paraventricular nucleus of the hypothalamus (PVN) and in the rostral ventrolateral medulla (RVLM) to suppress neuronal excitability, resulted in a long term decrease of blood pressure and sympathetic output in spontaneously hypertensive rats (SHR). OBJECTIVE: Evaluate gene expression in end-organs of SHR after a chronic overexpression of hKir2.1 channels in either the PVN or RVLM. METHODS: mRNA levels of 16 genes known to be involved with blood pressure regulation were evaluated using RT-PCR in tissues from the heart, common carotid artery and kidney of SHR submitted to chronic depression of PVN and RVLM excitability using a lentiviral vector (LVhKir2.1). RESULTS: In SHR hearts in which either the PVN or RVLM were injected with LVhKir2.1, there was a downregulation of angiotensin II receptor 1b (AT1), ATPase, Ca(2+)-transporter, troponin T2 and tropomyosin2 (only in RVLM) relative to the sham group. In the kidney of SHR with LVhKir2.1 injections in PVN and RVLM, angiotensinogen, angiotensin II receptor2 (AT2) and endothelin1 were all upregulated compared to sham. In the carotid artery, endothelin2, endothelin receptor A and B were up-regulated following LVhKir2.1 in to either the PVN or RVLM relative to sham. CONCLUSION: Chronic overexpression of hKir2.1 channels in PVN and RVLM, promoted a BP decrease with up-regulation of angiotensinogen and AT2 genes expression in the kidney and down-regulation of AT1 in the heart of SHR. Thus, we demonstrate the potential efficacy of central manipulation to protect against end-organ damage in essential hypertension.

Lead toxicity promotes autonomic dysfunction with increased chemoreceptor sensitivity.

Mortality and morbidity by toxic metals is an important issue of occupational health. Lead is an ubiquitous heavy metal in our environment despite having no physiological role in biological systems. Being an homeostatic controller is expected that the autonomic nervous system would show a degree of impairment in lead toxicity. In fact, sympathoexcitation associated to high blood pressure and tachypnea has been described together with baroreflex dysfunction. However, the mechanisms underlying the autonomic dysfunction and the interplay between baro- and chemoreflex are not yet fully clarified. The angiotensinogenic PVN-NTS axis (paraventricular nucleus of the hypothalamus - nucleus tractus solitarius axis) is a particularly important neuronal pathway that could be responsible for the autonomic dysfunction and the cardiorespiratory impairment in lead toxicity. Within the current work, we addressed in vivo, baro- and chemoreceptor reflex behaviour, before and after central angiotensin inhibition, in order to better understand the cardiorespiratory autonomic mechanisms underlying the toxic effects of long-term lead exposure. For that, arterial pressure, heart rate, respiratory rate, sympathetic and parasympathetic activity and baro- and chemoreceptor reflex profiles of anaesthetized young adult rats exposed to lead, from foetal period to adulthood, were evaluated. Results showed increased chemosensitivity together with baroreceptor reflex impairment, sympathetic over-excitation, hypertension and tachypnea. Chemosensitivity and sympathetic overexcitation were reversed towards normality values by NTS treatment with A-779, an angiotensin (1-7) antagonist. No parasympathetic changes were observed before and after A-799 treatment. In conclusion, angiotensin (1-7) at NTS level is involved in the autonomic dysfunction observed in lead toxicity. The increased sensitivity of chemoreceptor reflex expresses the clear impairment of autonomic outflow to the cardiovascular and respiratory systems induced by putative persistent, long duration, alert reaction evoked by the long term exposure to lead toxic effects. The present study brings new insights on the central mechanisms implicated in the autonomic dysfunction induced by lead exposure which are relevant for the development of additional therapeutic options to tackle lead toxicity symptoms.

Essential role of RVL medullary neuronal activity in the long term maintenance of hypertension in conscious SHR.

BACKGROUND: It is well established that sympathetic nervous system is responsible for the onset, development and maintenance of neurogenic hypertension. The rostroventrolateral medulla (RVLM) and medullo-cervical pressor area (MCPA) are important central sympathoexcitatory regions whose role on neurogenic hypertension remains unknown. OBJECTIVE: To establish RVLM and MCPA roles in the long-term regulation of blood pressure by depressing their neuron activity through the over-expression of hKir2.1-potassium channel in conscious spontaneously hypertensive rats (SHR). METHODS: In SHR, a lentiviral vector LVV-hKir2.1 was microinjected into RVLM or MCPA areas. A sham group was injected with LVV-eGFP. Blood pressure (BP) and heart rate (HR) were continuously monitored for 75 days. Baroreflex and chemoreflex functions were evaluated. Baroreflex gain, chemoreflex sensitivity, BP and HR variability were calculated. RESULTS: LVV-hKir2.1 expression in RVLM, but not in MCPA, produced a significant time-dependent decrease in systolic, diastolic, mean-BP and LF of systolic BP at 60-days post-injection. No significant changes were seen in LVV-eGFP RVLM injected SHR. CONCLUSION: Data show that chronic expression of Kir2.1 in the RVLM of conscious SHR caused a marked and sustained decrease in BP without changes in the baro- and peripheral chemoreceptor reflex evoked responses. This decrease was mostly due to a reduction in sympathetic output revealed indirectly by a decrease in the power density of the SBP-LF band. Our data are amongst the firsts to demonstrate the role of the RVLM in maintaining BP levels in hypertension in conscious SHR. We suggest that a decrease in RVLM neuronal activity is an effective anti-hypertensive treatment strategy.

Chronic depression of hypothalamic paraventricular neuronal activity produces sustained hypotension in hypertensive rats.

Changes in the sympathetic nervous system are responsible for the initiation, development and maintenance of hypertension. An important central sympathoexcitatory region is the paraventricular nucleus (PVN) of the hypothalamus, which may become more active in hypertensive conditions, as shown in acute studies previously. Our objective was to depress PVN neuronal activity chronically by the overexpression of an inwardly rectifying potassium channel (hKir2.1), while evaluating the consequences on blood pressure (BP) and its reflex regulation. In spontaneously hypertensive rats (SHRs) and Wistar rats (WKY) lentiviral vectors (LVV-hKir2.1; LV-TREtight-Kir-cIRES-GFP5 4 × 10(9) IU and LV-Syn-Eff-G4BS-Syn-Tetoff 6.2 × 10(9) IU in a ratio 1:4) were stereotaxically microinjected bilaterally into the PVN. Sham-treated SHRs and WKY received bilateral PVN microinjections of LVV-eGFP (LV-Syn-Eff-G4BS-Syn-Tetoff 6.2 × 10(9) IU and LV-TREtight-GFP 5.7 × 10(9) IU in a ratio 1:4). Blood pressure was monitored continuously by radio-telemetry and evaluated over 75 days. Baroreflex gain was evaluated using phenylephrine (25 µg ml(-1), i.v.), whereas lobeline (25 µg ml(-1), i.v.) was used to stimulate peripheral chemoreceptors. In SHRs but not normotensive WKY rats, LVV-hKir2.1 expression in the PVN produced time-dependent and significant decreases in systolic (from 158 ± 3 to 132 ± 6 mmHg; P < 0.05) and diastolic BP (from 135 ± 4 to 113 ± 5 mmHg; P < 0.05). The systolic BP low-frequency band was reduced (from 0.79 ± 0.13 to 0.42 ± 0.09 mmHg(2); P < 0.05), suggesting reduced sympathetic vasomotor tone. Baroreflex gain was increased and peripheral chemoreflex depressed after PVN microinjection of LVV-hKir2.1. We conclude that the PVN plays a major role in long-term control of BP and sympathetic nervous system activity in SHRs. This is associated with reductions in both peripheral chemosensitivity and respiratory-induced sympathetic modulation and an improvement in baroreflex sensitivity. Our results support the PVN as a powerful site to control BP in neurogenic hypertension.