A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

Design and development of cationic liposomes as DNA vaccine adjuvants

Cationic liposomes have been extensively explored for their efficacy in delivering nucleic acids, by offering the ability to protect plasmid DNA against degradation, promote gene expression and, in the case of DNA vaccines, induce both humoural and cellular immune responses. DNA vaccines may also offer advantages in terms of safety, but they are less effective and need an adjuvant to enhance their immunogenicity. Therefore, cationic liposomes can be utilised as delivery systems and/or adjuvants for DNA vaccines to stimulate stronger immune responses. To explore the role of liposomal systems within plasmid DNA delivery, parameters such as the effect of lipid composition, method of liposome preparation and presence of electrolytes in the formulation were investigated in characterisation studies, in vitro transfection studies and in vivo biodistribution and immunisation studies. Liposomes composed of 1,2-dioleoyl-sn-glycero 3-phosphoethanolamine (DOPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3- trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method and hydrated in aqueous media with or without presence of electrolytes. Whilst the in vitro transfection efficiency of all liposomes resulted to be higher than Lipofectin, DSTAP-based liposomes showed significantly higher transfection efficiency than DOTAP-based formulations. Furthermore, upon intramuscular injection of liposomal DNA vaccines, DSTAP-based liposomes showed a significantly stronger depot effect at the injection site. This could explain the result of heterologous immunisation studies, which revealed DSTAP-based liposomal vaccines induce stronger immune responses compared to DOTAP-based formulations. Previous studies have shown that having more liposomally associated antigen at the injection site would lead to more drainage of them into the local lymph nodes. Consequently, this would lead to more antigens being presented to antigen presenting cells, which are circulating in lymph nodes, and this would initiate a stronger immune response. Finally, in a comparative study, liposomes composed of dimethyldioctadecylammonium bromide (DDA) in combination with DOPE or immunostimulatory molecule of trehalose 6,6-dibehenate (TDB) were prepared and investigated in vitro and in vivo. Results showed that although DDA:TDB is not able to transfect the cells efficiently in vitro, this formulation induces stronger immunity compared to DDA:DOPE due to the immunostimulatory effects of TDB. This study demonstrated, while the presence of electrolytes did not improve immune responses, small unilamellar vesicle (SUV) liposomes induced stronger humoural immune responses compared to dehydration rehydration vesicle (DRV) liposomes. Moreover, lipid composition was shown to play a key role in in vitro and in vivo behaviour of the formulations, as saturated cationic lipids provided stronger immune responses compared to unsaturated lipids. Finally, heterologous prime/boost immunisation promoted significantly stronger immune responses compared to homologous vaccination of DNA vaccines, however, a single immunisation of subunit vaccine provoked comparable levels of immune response to the heterologous regimen, suggesting more immune efficiency for subunit vaccines compared to DNA vaccines.

Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies. © 2010 by the authors.

VaxiJen:a server for prediction of protective antigens, tumour antigens and subunit vaccines

Background - Vaccine development in the post-genomic era often begins with the in silico screening of genome information, with the most probable protective antigens being predicted rather than requiring causative microorganisms to be grown. Despite the obvious advantages of this approach – such as speed and cost efficiency – its success remains dependent on the accuracy of antigen prediction. Most approaches use sequence alignment to identify antigens. This is problematic for several reasons. Some proteins lack obvious sequence similarity, although they may share similar structures and biological properties. The antigenicity of a sequence may be encoded in a subtle and recondite manner not amendable to direct identification by sequence alignment. The discovery of truly novel antigens will be frustrated by their lack of similarity to antigens of known provenance. To overcome the limitations of alignment-dependent methods, we propose a new alignment-free approach for antigen prediction, which is based on auto cross covariance (ACC) transformation of protein sequences into uniform vectors of principal amino acid properties. Results - Bacterial, viral and tumour protein datasets were used to derive models for prediction of whole protein antigenicity. Every set consisted of 100 known antigens and 100 non-antigens. The derived models were tested by internal leave-one-out cross-validation and external validation using test sets. An additional five training sets for each class of antigens were used to test the stability of the discrimination between antigens and non-antigens. The models performed well in both validations showing prediction accuracy of 70% to 89%. The models were implemented in a server, which we call VaxiJen. Conclusion - VaxiJen is the first server for alignment-independent prediction of protective antigens. It was developed to allow antigen classification solely based on the physicochemical properties of proteins without recourse to sequence alignment. The server can be used on its own or in combination with alignment-based prediction methods.

Identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties

Subunit vaccine discovery is an accepted clinical priority. The empirical approach is time- and labor-consuming and can often end in failure. Rational information-driven approaches can overcome these limitations in a fast and efficient manner. However, informatics solutions require reliable algorithms for antigen identification. All known algorithms use sequence similarity to identify antigens. However, antigenicity may be encoded subtly in a sequence and may not be directly identifiable by sequence alignment. We propose a new alignment-independent method for antigen recognition based on the principal chemical properties of protein amino acid sequences. The method is tested by cross-validation on a training set of bacterial antigens and external validation on a test set of known antigens. The prediction accuracy is 83% for the cross-validation and 80% for the external test set. Our approach is accurate and robust, and provides a potent tool for the in silico discovery of medically relevant subunit vaccines.

Improving T cell-induced response to subunit vaccines:opportunities for a proteomic systems approach

Prophylactic vaccines are an effective strategy to prevent development of many infectious diseases. With new and re-emerging infections posing increasing risks to food stocks and the health of the population in general, there is a need to improve the rationale of vaccine development. One key challenge lies in development of an effective T cell-induced response to subunit vaccines at specific sites and in different populations. Objectives: In this review, we consider how a proteomic systems-based approach can be used to identify putative novel vaccine targets, may be adopted to characterise subunit vaccines and adjuvants fully. Key findings: Despite the extensive potential for proteomics to aid our understanding of subunit vaccine nature, little work has been reported on identifying MHC 1-binding peptides for subunit vaccines generating T cell responses in the literature to date. Summary: In combination with predictive and structural biology approaches to mapping antigen presentation, proteomics offers a powerful and as yet un-tapped addition to the armoury of vaccine discovery to predict T-cell subset responses and improve vaccine design strategies.

Influenza vaccine effectiveness in Portugal: season 2015/2016 report

This report was prepared as part of the Project “Monitoring Influenza vaccine effectiveness during influenza seasons and pandemics in the European Union” and describes the results obtained in Portugal under the Protocol Agreement celebrated between EpiConcept SARL, Paris and National Health Institute Dr. Ricardo Jorge, Lisbon. Data and activities related to the individuals 65 years and more were funded by European Union’s Horizon 2020 research and innovation programme under grant agreement no 634446.

Efficacy of the herpes zoster zubunit vaccine in adults 70 years of age or older

Background: A trial involving adults 50 years of age or older (ZOE-50) showed that the herpes zoster subunit vaccine (HZ/su) containing recombinant varicella-zoster virus glycoprotein E and the AS01B adjuvant system was associated with a risk of herpes zoster that was 97.2% lower than that associated with placebo. A second trial was performed concurrently at the same sites and examined the safety and efficacy of HZ/su in adults 70 years of age or older (ZOE-70).

Methods: This randomized, placebo-controlled, phase 3 trial was conducted in 18 countries and involved adults 70 years of age or older. Participants received two doses of HZ/su or placebo (assigned in a 1:1 ratio) administered intramuscularly 2 months apart. Vaccine efficacy against herpes zoster and postherpetic neuralgia was assessed in participants from ZOE-70 and in participants pooled from ZOE-70 and ZOE-50.

Results: In ZOE-70, 13,900 participants who could be evaluated (mean age, 75.6 years) received either HZ/su (6950 participants) or placebo (6950 participants). During a mean follow-up period of 3.7 years, herpes zoster occurred in 23 HZ/su recipients and in 223 placebo recipients (0.9 vs. 9.2 per 1000 person-years). Vaccine efficacy against herpes zoster was 89.8% (95% confidence interval [CI], 84.2 to 93.7; P<0.001) and was similar in participants 70 to 79 years of age (90.0%) and participants 80 years of age or older (89.1%). In pooled analyses of data from participants 70 years of age or older in ZOE-50 and ZOE-70 (16,596 participants), vaccine efficacy against herpes zoster was 91.3% (95% CI, 86.8 to 94.5; P<0.001), and vaccine efficacy against postherpetic neuralgia was 88.8% (95% CI, 68.7 to 97.1; P<0.001). Solicited reports of injection-site and systemic reactions within 7 days after injection were more frequent among HZ/su recipients than among placebo recipients (79.0% vs. 29.5%). Serious adverse events, potential immune-mediated diseases, and deaths occurred with similar frequencies in the two study groups.

Conclusions: In our trial, HZ/su was found to reduce the risks of herpes zoster and postherpetic neuralgia among adults 70 years of age or older.

Towards in silico IBV vaccine design: defining the role of polymorphism in viral attenuation

The gammacoronavirus, Infectious Bronchitis Virus (IBV), is a respiratory pathogen of chickens. IBV is a constant threat to poultry production as established vaccines are often ineffective against emerging strains. This requires constant and rapid vaccine production by a process of viral attenuation by egg passage, but the essential forces leading to attenuation in the virus have not yet been characterised. Knowledge of these factors will lead to the development of more effective, rationally attenuated, live vaccines and reduction of the mortality and morbidity caused by this pathogen. M41 CK strain was egg passaged four times many years ago at Houghton Poultry Research Station and stored as M41-CK EP4 (stock virus at The Pirbright Institute since 1992). It was the first egg passage to have its genome pyrosequenced and was therefore used as the baseline reference. The overall aim of this project was to analyse deep sequence data obtained from four IBV isolates (called A, A1, C and D) each originating from the common M41-CK EP4 (ep4) and independently passaged multiple times in embryonated chicken eggs (figure 1.1). Highly polymorphic encoding regions of the IBV genome were then identified which are likely involved in the attenuation process through the formation of independent SNPs and/or SNP clusters. This was then used to direct targeted investigation of SNPs during the attenuation process of the four IBV passages. A previously generated deep sequence dataset was used as a preliminary map of attenuation for one virulent strain of IBV. This investigation showed the nucleocapsid and spike as two highly polymorphic encoding regions within the IBV genome with the highest proportion of SNPs compared to encoding region size. This analysis then led to more focussed studies of the nucleocapsid and spike encoding region with the ultimate aim of mapping key attenuating regions and nucleotide positions. The 454 pyrosequencing data and further investigation of nucleocapsid and spike encoding regions have identified the SNPs present at the same nucleotide positions within analysed A, A1, C and D isolates. These SNPs probably play a crucial role in viral attenuation and universal vaccine production but it is not clear if independent SNPs are also involved in loss of virulence. The majority of SNPs accumulated at different nucleotide positions without further continuation in Sanger sequenced egg passages presenting S2 subunit (spike) and nucleocapsid as polymorphic encoding regions which in nature remain highly conserved.

Experimental Infection of Culex Annulirostris, Culex Gelidus, and Aedes Vigilax With a Yellow Fever/Japanese Encephalitis Virus Vaccine Chimera (Chimerivax TM-JE)

Australian mosquitoes from which Japanese encephalitis virus (JEV) has been recovered (Culex annulirostris, Culex gelidus, and Aedes vigilax) were assessed for their ability to be infected with the ChimeriVax-JE vaccine, with yellow fever vaccine virus 17D (YF 17D) from which the backbone of ChimeriVax-JE vaccine is derived and with JEV-Nakayama. None of the mosquitoes became infected after being fed orally with 6.1 log(10) plaque-forming units (PFU)/mL of ChimeriVax-JE vaccine, which is greater than the peak viremia in vaccinees (mean peak viremia = 4.8 PFU/mL, range = 0-30 PFU/mL of 0.9 days mean duration, range = 0-11 days). Some members of all three species of mosquito became infected when fed on JEV-Nakayama, but only Ae. vigilax was infected when fed on YF 17D. The results suggest that none of these three species of mosquito are likely to set up secondary cycles of transmission of ChimeriVax-JE in Australia after feeding on a viremic vaccinee.

Persistent chlamydial infections : role in inflammation and challenges for vaccine development.

The development of chlamydial vaccines continues to be a major challenge for researchers. While acute infections are the main target of vaccine development groups, Chlamydia is well known for its ability to establish chronic or persistent infections in its host. To date, little effort has focussed specifically on the challenges of vaccines to successfully combat the chronic or persistent phase of the disease and yet this will be a necessary aspect of any fully successful chlamydial vaccine. This short review specifically examines the phenomenon of chlamydial persistence and the unique challenges that this brings to vaccine development.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

Long term immunity to live attenuated Japanese encephalitis chimeric virus vaccine : randomized, double-blind, 5-year phase II study in healthy adults.

In a randomized, double-blind study, 202 healthy adults were randomized to receive a live, attenuated Japanese encephalitis chimeric virus vaccine (JE-CV) and placebo 28 days apart in a cross-over design. A subgroup of 98 volunteers received a JE-CV booster at month 6. Safety, immunogenicity, and persistence of antibodies to month 60 were evaluated. There were no unexpected adverse events (AEs) and the incidence of AEs between JE-CV and placebo were similar. There were three serious adverse events (SAE) and no deaths. A moderately severe case of acute viral illness commencing 39 days after placebo administration was the only SAE considered possibly related to immunization. 99% of vaccine recipients achieved a seroprotective antibody titer ≥ 10 to JE-CV 28 days following the single dose of JE-CV, and 97% were seroprotected at month 6. Kaplan Meier analysis showed that after a single dose of JE-CV, 87% of the participants who were seroprotected at month 6 were still protected at month 60. This rate was 96% among those who received a booster immunization at month 6. 95% of subjects developed a neutralizing titer ≥ 10 against at least three of the four strains of a panel of wild-type Japanese encephalitis virus (JEV) strains on day 28 after immunization. At month 60, that proportion was 65% for participants who received a single dose of JE-CV and 75% for the booster group. These results suggest that JE-CV is safe, well tolerated and that a single dose provides long-lasting immunity to wild-type strains

Concomitant or sequential administration of live attenuated Japanese encephalitis chimeric virus vaccine and yellow fever 17D vaccine : randomized double-blind phase II evaluation of safety and immunogenicity

A randomized, double-blind, study was conducted to evaluate the safety, tolerability and immunogenicity of a live attenuated Japanese encephalitis chimeric virus vaccine (JE-CV) co-administered with live attenuated yellow fever (YF) vaccine (YF-17D strain; Stamaril(®), Sanofi Pasteur) or administered successively. Participants (n = 108) were randomized to receive: YF followed by JE-CV 30 days later, JE followed by YF 30 days later, or the co-administration of JE and YF followed or preceded by placebo 30 days later or earlier. Placebo was used in a double-dummy fashion to ensure masking. Neutralizing antibody titers against JE-CV, YF-17D and selected wild-type JE virus strains was determined using a 50% serum-dilution plaque reduction neutralization test. Seroconversion was defined as the appearance of a neutralizing antibody titer above the assay cut-off post-immunization when not present pre-injection at day 0, or a least a four-fold rise in neutralizing antibody titer measured before the pre-injection day 0 and later post vaccination samples. There were no serious adverse events. Most adverse events (AEs) after JE vaccination were mild to moderate in intensity, and similar to those reported following YF vaccination. Seroconversion to JE-CV was 100% and 91% in the JE/YF and YF/JE sequential vaccination groups, respectively, compared with 96% in the co-administration group. All participants seroconverted to YF vaccine and retained neutralizing titers above the assay cut-off at month six. Neutralizing antibodies against JE vaccine were detected in 82-100% of participants at month six. These results suggest that both vaccines may be successfully co-administered simultaneously or 30 days apart.