Citrulline protects mice from experimental cerebral malaria by ameliorating hypoargininemia, urea cycle changes and vascular leak.

Clinical and model studies indicate that low nitric oxide (NO) bioavailability due in part to profound hypoargininemia contributes to cerebral malaria (CM) pathogenesis. Protection against CM pathogenesis may be achieved by altering the diet before infection with Plasmodium falciparum infection (nutraceutical) or by administering adjunctive therapy that decreases CM mortality (adjunctive therapy). This hypothesis was tested by administering citrulline or arginine in experimental CM (eCM). We report that citrulline injected as prophylaxis immediately post infection (PI) protected virtually all mice by ameliorating (i) hypoargininemia, (ii) urea cycle impairment, and (iii) disruption of blood brain barrier. Citrulline prophylaxis inhibited plasma arginase activity. Parasitemia was similar in citrulline- and vehicle control-groups, indicating that protection from pathogenesis was not due to decreased parasitemia. Both citrulline and arginine administered from day 1 PI in the drinking water significantly protected mice from eCM. These observations collectively indicate that increasing dietary citrulline or arginine decreases eCM mortality. Citrulline injected ip on day 4 PI with quinine-injected ip on day 6 PI partially protected mice from eCM; citrulline plus scavenging of superoxide with pegylated superoxide dismutase and pegylated catalase protected all recipients from eCM. These findings indicate that ameliorating hypoargininemia with citrulline plus superoxide scavenging decreases eCM mortality.

Meta-Analysis and Potential Role of Preexisting Heterosubtypic Cellular Immunity Based on Variations in Disease Severity Outcomes for Influenza Live Viral Challenges in Humans.

Influenza live viral challenges in humans are valuable models for testing the efficacy of vaccines and antiviral agents. Volunteers are treated with an investigational agent, and their clinical outcomes postchallenge are compared to those of placebo-treated volunteers. Despite using a common protocol, similar recruitment criteria, and similar doses of the same challenge strain, we noticed differences in disease severity outcomes between the placebo groups from different studies. We investigated whether these differences were significant and, if so, whether any pattern and its possible causes could be identified. We compared the clinical outcomes postchallenge in placebo groups from five clinical studies carried out between 2008 and 2013. Correlations between the prechallenge heterosubtypic cellular response (gamma interferon [IFN-γ]) and postchallenge clinical outcomes were also investigated in one study. Placebo groups from studies carried out between 2009 and 2010 attained significantly reduced (P < 0.05) symptom scores postchallenge compared to those of placebo groups from studies carried out in either 2008 or 2013. Also, in a 2010 study, the frequency of high-influenza heterosubtypic cellular responders prevaccination was significantly lower in the test group (FLU-v) than that in the placebo group (P = 0.04). Moreover, the increased preexisting heterosubtypic cellular response of the placebo group correlated with reductions in symptom score and viral shedding postchallenge (P ≤ 0.023). Only postvaccination did the test group display an equivalent correlation. The last influenza pandemic coincided with a significant reduction in disease severity outcomes. This reduction also appears to correlate with increased preexisting influenza heterosubtypic cellular responses. (This study is registered at ClinicalTrials.gov under registration number NCT01226758.).

A Synthetic Influenza Virus Vaccine Induces a Cellular Immune Response That Correlates with Reduction in Symptomatology and Virus Shedding in a Randomized Phase Ib Live-Virus Challenge in Humans.

Current influenza vaccines elicit primarily antibody-based immunity. They require yearly revaccination and cannot be manufactured until the identification of the circulating viral strain(s). These issues remain to be addressed. Here we report a phase Ib trial of a vaccine candidate (FLU-v) eliciting cellular immunity. Thirty-two males seronegative for the challenge virus by hemagglutination inhibition assay participated in this single-center, randomized, double-blind study. Volunteers received one dose of either the adjuvant alone (placebo, n = 16) or FLU-v (500 µg) and the adjuvant (n = 16), both in saline. Twenty-one days later, FLU-v (n = 15) and placebo (n = 13) volunteers were challenged with influenza virus A/Wisconsin/67/2005 (H3N2) and monitored for 7 days. Safety, tolerability, and cellular responses were assessed pre- and postvaccination. Virus shedding and clinical signs were assessed postchallenge. FLU-v was safe and well tolerated. No difference in the prevaccination FLU-v-specific gamma interferon (IFN-γ) response was seen between groups (average ± the standard error of the mean [SEM] for the placebo and FLU-v, respectively, 1.4-fold ± 0.2-fold and 1.6-fold ± 0.5-fold higher than the negative-control value). Nineteen days postvaccination, the FLU-v group, but not the placebo group, developed FLU-v-specific IFN-γ responses (8.2-fold ± 3.9-fold versus 1.3-fold ± 0.1-fold higher than the negative-control value [average ± SEM] for FLU-v versus the placebo [P = 0.0005]). FLU-v-specific cellular responses also correlated with reductions in both viral titers (P = 0.01) and symptom scores (P = 0.02) postchallenge. Increased cellular immunity specific to FLU-v correlates with reductions in both symptom scores and virus loads. (This study has been registered at ClinicalTrials.gov under registration no. NCT01226758 and at hra.nhs.uk under EudraCT no. 2009-014716-35.).

Inhibition of cough reflex sensitivity by diphenhydramine during acute viral respiratory tract infection.

BACKGROUND: Currently available over-the-counter cough remedies historically have been criticized for lack of scientific evidence supporting their efficacy. Although the first-generation antihistamine diphenhydramine is classified as an antitussive by the United States Food and Drug Administration, to the authors' knowledge it has never been shown to inhibit cough reflex sensitivity in subjects with pathological cough. OBJECTIVE: To evaluate the effect of diphenhydramine on cough reflex sensitivity. SETTING: Montefiore Medical Center, an academic medical center in New York City. METHODS: Twenty two subjects with acute viral upper respiratory tract infection (common cold) underwent cough reflex sensitivity measurement employing capsaicin challenge on 3 separate days, 2 h after ingesting single doses of study drug (to coincide with peak blood concentrations), administered in randomized, double-blind manner: a multicomponent syrup containing diphenhydramine (25 mg), phenylephrine (10 mg), in a natural cocoa formulation; dextromethorphan (30 mg) syrup; and, placebo syrup. The standard endpoint of cough challenge was used: concentration of capsaicin inducing ≥5 coughs (C5). MAIN OUTCOME MEASURE: Effect on cough reflex sensitivity (C5). RESULTS: A significant difference (p = 0.0024) was established among groups, with pairwise analysis revealing a significant increase in mean log C5 (0.4 ± 0.55 (SD); p < 0.01) for the diphenhydramine-containing medication versus placebo, but not for dextromethorphan versus placebo. CONCLUSIONS: Our results provide the initial evidence of the ability of diphenhydramine to inhibit cough reflex sensitivity in subjects with acute pathological cough. Timing of cough reflex sensitivity measurement may not have allowed demonstration of maximal antitussive effect of dextromethorphan.

Safety, immunogenicity and efficacy assessment of HIV immunotherapy in a multi-centre, double-blind, randomised, Placebo-controlled Phase Ib human trial.

BACKGROUND: Combination antiretroviral therapy (cART) is the main therapeutic management tool for HIV/AIDS. Despite its success in controlling viral load and disease progression, cART is expensive, associated with a range of significant side effects and depends for its efficacy on the patient's life-long commitment to high levels of treatment adherence. Immunotherapeutic agents can provide potential solutions to these shortcomings. Here we describe a Phase Ib trial of HIV-v, a synthetic immunotherapy that elicits T- and B-cell effector responses against HIV infected cells. METHODS: Fifty-nine cART-naive HIV-infected males aged 18-50 years with viral load of 5000-500,000 copies/ml and CD4 counts >350/µl were recruited for this multi-centre, randomised, double blind study. Volunteers received one low (250 µg) or high (500 µg) dose of HIV-v, either alone or adjuvanted (ISA-51). Safety, immunogenicity, CD4 count and viral load were monitored over 168 Days. RESULTS: HIV-v was well tolerated and the adjuvanted formulations elicited IgG responses in up to 75% of volunteers. The high adjuvanted dose also elicited cellular responses in 45% of tested volunteers. In these responding subjects viral loads were reduced by over 1 log (p=0.04) compared to Placebo and non-responders. No changes in CD4 count were observed. CONCLUSIONS: HIV-v is safe and can elicit T- and B-cell responses in ART-naive HIV patients that significantly reduce viral load. Improved dosing regimens and further research on long term efficacy are required, but HIV-v appears to have potential as an immunotherapeutic anti-viral agent. Trial registered as EudraCT-2009-010593-37 (ClinicalTrials.gov Identifier: NCT01071031).

Synthetic immunotherapy induces HIV virus specific Th1 cytotoxic response and death of an HIV-1 infected human cell line through classic complement activation.

BACKGROUND: This manuscript describes the development of a novel synthetic immunotherapy (HIV-v) composed of four multi-epitope polypeptides targeting conserved regions in the Nef, Rev, Vif and Vpr viral proteins. Immunogenicity and cytotoxicity of HIV-v are discussed. METHODS: Short conserved T-cell multi-epitope regions were identified in silico in the HIV proteome. The immunogenicity of the identified HIV-v polypeptides was assessed in vivo by immunisation of C57BLK6 mice transgenic for HLA-A*0201. Splenocytes from immunised animals were exposed in vitro to soluble HIV-v polypeptides or to syngeneic (T1) or allogeneic (Jurkat) cells transfected with these polypeptides. Specific T-cell reactivity was assessed by cell-based IFN-γ ELISA. Virus specific CD3 + CD8+ IFN-γ+ recall responses were also determined by flow cytometry following in vitro exposure of splenocytes from immunised mice to syngeneic (T1) and allogeneic (H9) cells infected with HIV-1 strain IIIB. HIV-v specific antibodies were quantified by ELISA whilst antibody mediated anti-viral immunotherapeutic effect on T1 cells infected with a laboratory adapted and a primary isolate of the HIV-1 virus was assessed in a LDH-based complement mediated lysis assay. RESULTS: HIV-v elicited antigen-specific IgG and IFN-γ responses against the synthetic polypeptides in the formulation. HIV-v specific T cells recognised polypeptides presented either as soluble antigen or complexed to HLA-A*0201 following natural processing and presentation by syngeneic human T1 cells. Moreover, the CD3 + CD8+ component of the response recognised syngeneic T1 cells naturally infected with HIV-1 in a virus-specific and MHC restricted-manner. The HIV-v specific IgG response was also able to recognise human T1 cells naturally infected with HIV-1 and induce cell death through classic activation of complement. CONCLUSIONS: HIV-v induces a vaccine-specific type I immune response characterised by activation of effector CD8+ T cell and antibody responses that recognise and kill human cell lines naturally infected with a laboratory adapted and a primary isolate of the HIV-1 virus. The data supports the hypothesis that alternative HIV protein targets can be effectively used to prime both cellular and antibody immune responses of clinical value in the prevention and treatment of HIV infection.

Synthetic Influenza vaccine (FLU-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled Phase I trial.

OBJECTIVES: Current Influenza vaccines elicit antibody mediated prophylactic immunity targeted to viral capsid antigens. Despite their global use these vaccines must be administered yearly to the population, cannot be manufactured until the circulating viral strain(s) have been identified and have limited efficacy. A need remains for Influenza vaccines addressing these issues and here we report the results of a Phase Ib trial of a novel synthetic Influenza vaccine (FLU-v) targeting T cell responses to NP, M1 and M2. METHODS: Forty-eight healthy males aged 18-40 were recruited for this single-centre, randomised, double blind study. Volunteers received one single low (250 µg) or high (500 µg) dose of FLU-v, either alone or adjuvanted. Safety, tolerability and basic immunogenicity (IgG and IFN-γ responses) parameters were assessed pre-vaccination and for 21 days post-vaccination. RESULTS: FLU-v was found to be safe and well tolerated with no vaccine associated severe adverse events. Dose-dependent IFN-γ responses >2-fold the pre-vaccination level were detected in 80% and 100% of volunteers receiving, respectively, the low and high dose adjuvanted FLU-v formulations. No formulation tested induced any significant FLU-v antibody response. CONCLUSION: FLU-v is safe and induces a vaccine-specific cellular immunity. Cellular immune responses are historically known to control and mitigate infection and illness during natural infection.

Synthetic multi-epitope peptides identified in silico induce protective immunity against multiple influenza serotypes.

Influenza causes yearly epidemics of mild disease and, occasionally, pandemics with millions of fatalities. Currently, no vaccine is effective against all influenza strains. Analysis of influenza sequences from animal and human isolates using CLUSTALW and a novel proprietary epitope prediction algorithm identified six conserved T cell-reactive regions in several proteins. Immunisation of transgenic mice with a preparation of these six regions as chemically synthesised peptides (FLU-v) induced a specific HLA-A*0201-mediated CD8(+) T cell response. This T cell population also reacted against human cells infected with three non-related influenza strains, confirming that the identified regions contain epitopes naturally presented by infected human cells and conserved amongst non-related viruses. Moreover, FLU-v immunisation significantly increased survival of transgenic mice against lethal challenge with influenza. Overall, FLU-v represents a promising influenza vaccine candidate, obviating the need for yearly vaccinations and allowing the stockpiling and initiation of a worldwide vaccination program in advance of a pandemic outbreak.

Effect of dose and long-term storage on the immunogenicity of murine polyomavirus VP1 virus-like particles.

We have analysed the stability and immunogenicity of murine polyomavirus virus-like particles (VLPs) following intranasal administration without adjuvant. No morphological or immunological changes were observed in a preparation of these VLPs stored for 9 weeks at room temperature. Strong humoral and cellular (Th1) responses were obtained after a single 5.55 microg dose immunisation, which are efficiently boosted after a second dose. However, at dose concentrations above 0.22 microg/microl, these VLPs appear to aggregate and, when used for immunisations, they fail to induce a strong cellular response, even though the humoral response is unaffected. These results may reflect the differential processing of VLP aggregates by the immune system or, alternatively, VLP neutralisation by antibodies induced after a primary immunisation.

Enhanced tumour growth after DNA vaccination against human papilloma virus E7 oncoprotein: evidence for tumour-induced immune deviation.

We have examined the induction of anti-tumour immunity in a murine model using a gene vaccine approach to deliver a well defined tumour antigen. The vaccines expressed the human papilloma virus type 16 (HPV 16) E7 oncoprotein, and protection was measured against HPV 16-expressing C3R tumour cell line in vivo. In control mice injected with saline, C3R cells initially formed tumours but then regressed completely. As expected, animals injected with a peptide that represents the D(b)-presented CTL epitope from E7 (RAHYNIVTF) were completely protected from tumour growth. Contrary to expectation, however, we consistently saw enhanced tumour growth, delayed regression, or tumour outgrowth in mice vaccinated with two different E7-expressing DNA vaccines. We found no evidence for loss of D(b) or K(b) class I MHC molecules from C3R cells recovered from outgrown tumours, and fluorescent MHC/peptide tetramer staining revealed E7 gene vaccination did not delete RAHYNIVTF-specific CD8(+) T cells. However, we did observe an effect on cytokine production. Splenocytes from E7 gene vaccinated animals responded to re-stimulation in vitro with C3R cells by producing IL-4 but background levels of IFN-gamma. We also observed that cytokine production and E7 peptide-specific CTL were only detectable in vaccinated animals after C3R challenge, but not after DNA priming alone. We conclude that 'prime-boosting' is necessary to observe tumour-specific T cell responses with the gene vaccine approach, but that boosting with tumour cells causes skewing of the primed cells in a T2 direction that is incompatible with protective anti-tumour immunity.

A role for liposomes in genetic vaccination.

Genetic immunization by the use of plasmid DNA encoding antigens from bacteria, viruses, protozoa and cancers has often led to protective humoral and cell-mediated immunity, and has some practical advantages over conventional vaccines. However, naked DNA vaccines can be degraded by nucleases in situ, are unable to target antigen presenting cells (APCs), and exhibit poor performance when administered by routes other than the intramuscular, all of which have reduced the value of the approach. We have been able to avoid DNA degradation and also target DNA to APCs by the use of liposomes as DNA vaccine carriers. Entrapment of plasmid DNA within the aqueous spaces of cationic liposomes is effected by a one step procedure which results in most of the DNA being incorporated into a freeze dried, ready to use preparation. Animal experiments have shown that immunization by the intramuscular or the subcutaneous route with liposome-entrapped plasmid DNA encoding the hepatitis B surface antigen leads to much greater humoral (IgG subclasses) and cell mediated (splenic IFN-gamma) immune responses than with naked DNA. In other experiments with a plasmid DNA encoding a model antigen (ovalbumin), a cytotoxic T lymphocyte (CTL) response was also observed. These results could be explained by the ability of liposomes to protect their DNA content from local nucleases and direct it to APCs in the lymph nodes draining the injected site.

Induction of a cytotoxic T lymphocyte (CTL) response to plasmid DNA delivered via Lipodine liposomes.

We have previously shown that liposome-mediated plasmid DNA immunisation may be a preferred alternative to the use of naked DNA. Lipodine DNA formulations consist of liposomes containing entrapped DNA plasmid by the dehydration-rehydration (DRV) method. Such liposome formulations are distinct from liposomes with externally complexed DNA in that the majority of the DNA is "internal" to the liposome structure and hence protected from DNAase degradation. Previous studies on the immune response induced by DNA vaccines entrapped in Lipodine have focused on the humoural response. In the present study, we have expanded the analysis profile in order to include the cytotoxic T lymphocyte (CTL) component of the immune response. We have analysed the immune response induced by DNA entrapped in Lipodine compared to that induced by DNA alone when delivered subcutaneously, a route of administration not normally inducing significant plasmid DNA mediated immune activation. Our results indicate that delivery of a small dose of plasmid DNA in Lipodine results in an improved antibody response to the plasmid encoded antigen and a strong antigen specific CTL response compared to that induced by DNA delivered alone.