Publications by authors named "Nicolas Collin"

38 Publications

Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters.

bioRxiv 2021 Mar 4. Epub 2021 Mar 4.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.
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http://dx.doi.org/10.1101/2021.03.03.433558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941618PMC
March 2021

H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets.

NPJ Vaccines 2020 May 11;5(1):38. Epub 2020 May 11.

Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands.

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation.
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http://dx.doi.org/10.1038/s41541-020-0187-4DOI Listing
May 2020

The Immunogenicity of Capsid-Like Particle Vaccines in Combination with Different Adjuvants Using Different Routes of Administration.

Vaccines (Basel) 2021 Feb 6;9(2). Epub 2021 Feb 6.

Centre for Medical Parasitology, Department of Immunology and Microbiology, University of Copenhagen, 1165 København, Denmark.

Capsid-like particle (CLP) displays can be used to enhance the immunogenicity of vaccine antigens, but a better understanding of how CLP vaccines are best formulated and delivered is needed. This study compared the humoral immune responses in mice elicited against two different vaccine antigens (a bacterial protein and a viral peptide) delivered on an AP205 CLP platform using six different adjuvant formulations. In comparison to antibody responses obtained after immunization with the unadjuvanted CLP vaccine, three of the adjuvant systems (neutral liposomes/monophosphoryl lipid A/quillaja saponaria 21, squalene-in-water emulsion, and monophosphoryl lipid A) caused significantly increased antibody levels, whereas formulation with the three other adjuvants (aluminum hydroxide, cationic liposomes, and cationic microparticles) resulted in similar or even decreased antibody responses. When delivering the soluble bacterial protein in a squalene-in-water emulsion, 4-log lower IgG levels were obtained compared to when the protein was delivered on CLPs without the adjuvant. The AP205 CLP platform promoted induction of both IgG1 and IgG2 subclasses, which could be skewed towards a higher production of IgG1 (aluminum hydroxide). Compared to other routes, intramuscular administration elicited the highest IgG levels. These results indicate that the effect of the external adjuvant does not always synergize with the adjuvant effect of the CLP display, which underscores the need for empirical testing of different extrinsic adjuvants.
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http://dx.doi.org/10.3390/vaccines9020131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915698PMC
February 2021

A Comparison of Intramuscular and Subcutaneous Administration of LigA Subunit Vaccine Adjuvanted with Neutral Liposomal Formulation Containing Monophosphoryl Lipid A and QS21.

Vaccines (Basel) 2020 Sep 1;8(3). Epub 2020 Sep 1.

Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.

Leptospirosis vaccines with higher potency and reduced adverse effects are needed for human use. The carboxyl terminal domain of leptospiral immunoglobulin like protein A (LigAc) is currently the most promising candidate antigen for leptospirosis subunit vaccine. However, LigAc-based vaccines were unable to confer sterilizing immunity against infection in animal models. Several factors including antigen properties, adjuvant, delivery system, and administration route need optimization to maximize vaccine efficacy. Our previous report demonstrated protective effects of the recombinant LigAc (rLigAc) formulated with liposome-based adjuvant, called LMQ (neutral liposome combined with monophosphoryl lipid A and fraction 21) in hamsters. This study aimed to evaluate the impact of two commonly used administration routes, intramuscular (IM) and subcutaneous (SC), on immunogenicity and protective efficacy of rLigAc-LMQ administrated three times at 2-week interval. Two IM vaccinations triggered significantly higher levels of total anti-rLigAc IgG than two SC injections. However, comparable IgG titers and IgG2/IgG1 ratio was observed for both routes after the third immunization. The route of vaccine administration did not influence the survival rate (60%) and renal colonization against lethal challenge. Importantly, the kidneys of IM group showed no pathological lesions while the SC group showed mild damage. In conclusion, IM vaccination with rLigAc-LMQ not only elicited faster antibody production but also protected from kidney damage following leptospiral infection better than SC immunization. However, both tested routes did not influence protective efficacy in terms of survival rate and the level of renal colonization.
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http://dx.doi.org/10.3390/vaccines8030494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565420PMC
September 2020

H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets.

NPJ Vaccines 2020 11;5:38. Epub 2020 May 11.

1Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands.

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation.
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http://dx.doi.org/10.1038/s41541-020-0187-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214439PMC
May 2020

Vaccine-Induced Th1-Type Response Protects against Invasive Group A Infection in the Absence of Opsonizing Antibodies.

mBio 2020 03 10;11(2). Epub 2020 Mar 10.

Australian Infectious Diseases Research Centre and School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia

Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-γ), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy. Availability of a group A vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.
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http://dx.doi.org/10.1128/mBio.00122-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064752PMC
March 2020

Efficacy of three innovative bacterin vaccines against experimental infection with Mycoplasma hyopneumoniae.

Vet Res 2019 Nov 8;50(1):91. Epub 2019 Nov 8.

Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820, Merelbeke, Belgium.

New vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective (RDS -61.90%, macroscopic lung lesions -88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) -67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.
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http://dx.doi.org/10.1186/s13567-019-0709-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842239PMC
November 2019

Reduced Renal Colonization and Enhanced Protection by Leptospiral Factor H Binding Proteins as a Multisubunit Vaccine Against Leptospirosis in Hamsters.

Vaccines (Basel) 2019 Aug 22;7(3). Epub 2019 Aug 22.

Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.

Subunit vaccines conferring complete protection against leptospirosis are not currently available. The interactions of factor H binding proteins (FHBPs) on pathogenic leptospires and host factor H are crucial for immune evasion by inhibition of complement-mediated killing. The inhibition of these interactions may be a potential strategy to clear leptospires in the host. This study aimed to evaluate a multisubunit vaccine composed of four known leptospiral FHBPs: LigA domain 7-13 (LigAc), LenA, LcpA, and Lsa23, for its protective efficacy in hamsters. The mono and multisubunit vaccines formulated with LMQ adjuvant, a combination of neutral iposome, onophosphoryl lipid A, and fraction 21, induced high and comparable specific antibody (IgG) production against individual antigens. Hamsters immunized with the multisubunit vaccine showed 60% survival following the challenge by 20 LD of serovar Pomona. No significant difference in survival rate and pathological findings of target organs was observed after vaccinations with multisubunit or mono-LigAc vaccines. However, the multisubunit vaccine significantly reduced leptospiral burden in surviving hamsters in comparison with the monosubunit vaccines. Therefore, the multisubunit vaccine conferred partial protection and reduced renal colonization against virulence infection in hamsters. Our multisubunit formulation could represent a promising vaccine against leptospirosis.
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http://dx.doi.org/10.3390/vaccines7030095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789851PMC
August 2019

Electroporation of a nanoparticle-associated DNA vaccine induces higher inflammation and immunity compared to its delivery with microneedle patches in pigs.

J Control Release 2019 08 29;308:14-28. Epub 2019 Jun 29.

VIM, INRA, Université Paris-Saclay, Domaine de Vilvert, 78350 Jouy-en-Josas, France. Electronic address:

DNA vaccination is an attractive technology, based on its well-established manufacturing process, safety profile, adaptability to rapidly combat pandemic pathogens, and stability at ambient temperature; however an optimal delivery method of DNA remains to be determined. As pigs are a relevant model for humans, we comparatively evaluated the efficiency of vaccine DNA delivery in vivo to pigs using dissolvable microneedle patches, intradermal inoculation with needle (ID), surface electroporation (EP), with DNA associated or not to cationic poly-lactic-co-glycolic acid nanoparticles (NPs). We used a luciferase encoding plasmid (pLuc) as a reporter and vaccine plasmids encoding antigens from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a clinically-significant swine arterivirus. Patches were successful at inducing luciferase expression in skin although at lower level than EP. EP induced the cutaneaous recruitment of granulocytes, of MHC2CD172A myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1β, IL-8 and IL-17; these local responses were more limited with ID and undetectable with patches. The addition of NP to EP especially promoted the recruitment of the MHC2CD172A CD163 and CD163 myeloid subsets. Notably we obtained the strongest and broadest IFNγ T-cell response against a panel of PRRSV antigens with DNA + NPs delivered by EP, whereas patches and ID were ineffective. The anti-PRRSV IgG responses were the highest with EP administration independently of NPs, mild with ID, and undetectable with patches. These results contrast with the immunogenicity and efficacy previously induced in mice with patches. This study concludes that successful DNA vaccine administration in skin can be achieved in pigs with electroporation and patches, but only the former induces local inflammation, humoral and cellular immunity, with the highest potency when NPs were used. This finding shows the importance of evaluating the delivery and immunogenicity of DNA vaccines beyond the mouse model in a preclinical model relevant to human such as pig and reveals that EP with DNA combined to NP induces strong immunogenicity.
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http://dx.doi.org/10.1016/j.jconrel.2019.06.041DOI Listing
August 2019

A DNA-Modified Live Vaccine Prime-Boost Strategy Broadens the T-Cell Response and Enhances the Antibody Response against the Porcine Reproductive and Respiratory Syndrome Virus.

Viruses 2019 06 14;11(6). Epub 2019 Jun 14.

VIM, INRA, Université Paris-Saclay, Domaine de Vilvert, 78350 Jouy-en-Josas, France.

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) induces reproductive disorders in sows and respiratory illnesses in growing pigs and is considered as one of the main pathogenic agents responsible for economic losses in the porcine industry worldwide. Modified live PRRSV vaccines (MLVs) are very effective vaccine types against homologous strains but they present only partial protection against heterologous viral variants. With the goal to induce broad and cross-protective immunity, we generated DNA vaccines encoding B and T antigens derived from a European subtype 1 strain that include T-cell epitope sequences known to be conserved across strains. These antigens were expressed either in a native form or in the form of vaccibodies targeted to the endocytic receptor XCR1 and CD11c expressed by different types of antigen-presenting cells (APCs). When delivered in skin with cationic nanoparticles and surface electroporation, multiple DNA vaccinations as a stand-alone regimen induced substantial antibody and T-cell responses, which were not promoted by targeting antigens to APCs. Interestingly, a DNA-MLV prime-boost strategy strongly enhanced the antibody response and broadened the T-cell responses over the one induced by MLV or DNA-only. The anti-nucleoprotein antibody response induced by the DNA-MLV prime-boost was clearly promoted by targeting the antigen to CD11c and XCR1, indicating a benefit of APC-targeting on the B-cell response. In conclusion, a DNA-MLV prime-boost strategy, by enhancing the potency and breadth of MLV vaccines, stands as a promising vaccine strategy to improve the control of PRRSV in infected herds.
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http://dx.doi.org/10.3390/v11060551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630347PMC
June 2019

Systems Immunology Characterization of Novel Vaccine Formulations for Bacterins.

Front Immunol 2019 24;10:1087. Epub 2019 May 24.

Institute of Virology and Immunology, Mittelhäusern, Switzerland.

We characterized five different vaccine candidates and a commercial vaccine in terms of safety, immunogenicity and using a systems vaccinology approach, with the aim to select novel vaccine candidates against . Seven groups of six -free piglets were primo- and booster vaccinated with the different experimental bacterin formulations, the commercial vaccine Hyogen® as a positive control or PBS as a negative control. The experimental bacterin was formulated with cationic liposomes + c-di-AMP (Lipo_AMP), cationic liposomes + Toll-like receptor (TLR) 2/1, TLR7, and TLR9 ligands (TLR ligands; Lipo_TLR), micro-particles + TLR ligands (PLGA_TLR), squalene-in-water emulsion + TLR ligands (SWE_TLR), or DDA:TDB liposomes (Lipo_DDA:TDB). Lipo_DDA:TDB and Lipo_AMP were the most potent in terms of serum antibody induction, and Lipo_DDA:TDB, Lipo_AMP, and SWE_TLR significantly induced Th1 cytokine-secreting T-cells. Only PLGA_TLR appeared to induce Th17 cells, but was unable to induce serum antibodies. The transcriptomic analyses demonstrated that the induction of inflammatory and myeloid cell blood transcriptional modules (BTM) in the first 24 h after vaccination correlated well with serum antibodies, while negative correlations with the same modules were found 7 days post-vaccination. Furthermore, many cell cycle and T-cell BTM upregulated at day seven correlated positively with adaptive immune responses. When comparing the delivery of the identical TLR ligands with the three formulations, we found SWE_TLR to be more potent in the induction of an early innate immune response, while the liposomal formulation more strongly promoted late cell cycle and T-cell BTM. For the PLGA formulation we found signs of a delayed and weak perturbation of these BTM. Lipo_AMP was found to be the most potent vaccine at inducing a BTM profile similar to that correlating with adaptive immune response in this and other studies. Taken together, we identified four promising vaccine candidates able to induce -specific antibody and T-cell responses. In addition, we have adapted a systems vaccinology approach developed for human to pigs and demonstrated its capacity in identifying early immune signatures in the blood relating to adaptive immune responses. This approach represents an important step in a more rational design of efficacious vaccines for pigs.
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http://dx.doi.org/10.3389/fimmu.2019.01087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543460PMC
September 2020

Equitable realization of the right to health in Haiti: how household data inform health seeking behavior and financial risk protection.

Int J Equity Health 2019 05 27;18(1):77. Epub 2019 May 27.

World Bank, Health Nutrition and Population (HNP), 1850 I St NW, Washington, DC, 20006, USA.

Background: Though the right to health is included in Haiti's constitution, little progress has been made to expand universal health coverage nationwide, a strategy to ensure access to health services for all, while preventing financial hardship among the poor. Realizing universal health coverage will require a better understanding of inequities in health care utilization and out-of-pocket payments for health. This study measures inequality in health services utilization and the determinants of health seeking behavior in Haiti. It also examines the determinants of catastrophic health expenditures, defined by the Sustainable Development Goal Framework (Indicator 3.8.2) as expenditures that exceed 10% of overall household expenditures.

Methodology: Three types of analysis were conducted using the 2012 and 2013 Household Surveys (Enquête sur les Conditions de Vie des Ménages Après Séisme (ECVMAS I (2012) and ECVMAS II (2013)) to measure: 1) outpatient services as a measure of inequalities using the 2013 Concentration Index; 2) drivers of health seeking behavior using a logistic regression model for 2013; and 3) determinants of catastrophic health expenditures using Seemingly Unrelated Regressions for both 2012 and 2013.

Results: The rate of catastrophic health expenditures increased nationwide from 9.43% in 2012 to 11.54% in 2013. This increase was most notable among the poorest wealth quintile (from 11.62% in 2012 to 18.20% in 2013), yet declined among the richest wealth quintile (from 9.49% to 4.46% during the same period). The increase in the rate of catastrophic health expenditures among the poorest coincides with a sharp decrease in external donor funding for the health sector. Regression analysis indicated that the rich wealth quintiles were less likely than poor wealth quintiles to incur catastrophic health expenditures. Interestingly, households were less likely to incur catastrophic health expenditures when they accessed care from Community Health Workers than when they received care from other types of providers, including public and private health care facilities. This study also shows that Community Health Worker-provided services have a negative concentration index (- 0.22) and are therefore most utilized by poor quintiles. In contrast, both public and private outpatient services had positive concentration indexes (0.05 and 0.12 respectively) and are most utilized by the rich wealth quintiles. Seeking care from traditional healers was found to be pro-poor in Haiti (concentration index of - 0.18) yet was also associated with higher catastrophic health expenditures albeit the coefficient was not significant.

Conclusion: The expansion of universal health coverage in Haiti is evolving in a 'pro-rich' manner. Realizing Haiti's right to health will require a course-correction supported by national policies that protect the poor wealth quintiles from catastrophic health expenditures. Such policies may include Community Health Worker service delivery expansion in underserved areas. Evidence-based interventions may also be required to lower outpatient user fees, subsidize drug costs and promote efficiencies in pro-poor disaster relief programming.
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http://dx.doi.org/10.1186/s12939-019-0973-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537186PMC
May 2019

A simian-adenovirus-vectored rabies vaccine suitable for thermostabilisation and clinical development for low-cost single-dose pre-exposure prophylaxis.

PLoS Negl Trop Dis 2018 10 29;12(10):e0006870. Epub 2018 Oct 29.

Jenner Institute, University of Oxford, Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom.

Background: Estimates of current global rabies mortality range from 26,000 to 59,000 deaths per annum. Although pre-exposure prophylaxis using inactivated rabies virus vaccines (IRVs) is effective, it requires two to three doses and is regarded as being too expensive and impractical for inclusion in routine childhood immunization programmes.

Methodology/ Principal Findings: Here we report the development of a simian-adenovirus-vectored rabies vaccine intended to enable cost-effective population-wide pre-exposure prophylaxis against rabies. ChAdOx2 RabG uses the chimpanzee adenovirus serotype 68 (AdC68) backbone previously shown to achieve pre-exposure protection against rabies in non-human primates. ChAdOx2 differs from AdC68 in that it contains the human adenovirus serotype 5 (AdHu5) E4 orf6/7 region in place of the AdC68 equivalents, enhancing ease of manufacturing in cell lines which provide AdHu5 E1 proteins in trans. We show that immunogenicity of ChAdOx2 RabG in mice is comparable to that of AdC68 RabG and other adenovirus serotypes expressing rabies virus glycoprotein. High titers of rabies virus neutralizing antibody (VNA) are elicited after a single dose. The relationship between levels of VNA activity and rabies virus glycoprotein monomer-binding antibody differs after immunization with adenovirus-vectored vaccines and IRV vaccines, suggesting routes to further enhancement of the efficacy of the adenovirus-vectored candidates. We also demonstrate that ChAdOx2 RabG can be thermostabilised using a low-cost method suitable for clinical bio-manufacture and ambient-temperature distribution in tropical climates. Finally, we show that a dose-sparing effect can be achieved by formulating ChAdOx2 RabG with a simple chemical adjuvant. This approach could lower the cost of ChAdOx2 RabG and other adenovirus-vectored vaccines.

Conclusions/ Significance: ChAdOx2 RabG may prove to be a useful tool to reduce the human rabies death toll. We have secured funding for Good Manufacturing Practice- compliant bio-manufacture and Phase I clinical trial of this candidate.
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http://dx.doi.org/10.1371/journal.pntd.0006870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224154PMC
October 2018

System immunology-based identification of blood transcriptional modules correlating to antibody responses in sheep.

NPJ Vaccines 2018 3;3:41. Epub 2018 Oct 3.

Institute of Virology and Immunology, Mittelhäusern, Switzerland.

Inactivated vaccines lack immunogenicity and therefore require potent adjuvants. To understand the in vivo effects of adjuvants, we used a system immunology-based analysis of ovine blood transcriptional modules (BTMs) to dissect innate immune responses relating to either antibody or haptoglobin levels. Using inactivated foot-and-mouth disease virus as an antigen, we compared non-adjuvanted to liposomal-formulated vaccines complemented or not with TLR4 and TLR7 ligands. Early after vaccination, BTM relating to myeloid cells, innate immune responses, dendritic cells, and antigen presentation correlated positively, whereas BTM relating to T and natural killer cells, as well as cell cycle correlated negatively with antibody responses. Interestingly, similar BTM also correlated with haptoglobin, but in a reversed manner, indicating that acute systemic inflammation is not beneficial for early antibody responses. Analysis of vaccine-dependent BTM modulation showed that liposomal formulations induced similar responses to those correlating to antibody levels. Surprisingly, the addition of the TLR ligands appeared to reduce early immunological perturbations and mediated anti-inflammatory effects, despite promoting antibody responses. When pre-vaccination BTM were analyzed, we found that high vaccine responders expressed higher levels of many BTM relating to cell cycle, antigen-presenting cells, and innate responses as compared with low responders. In conclusion, we have transferred human BTM to sheep and identified early vaccine-induced responses associated with antibody levels or unwanted inflammation in a heterogeneous and small group of animals. Such readouts are applicable to other veterinary species and very useful to identify efficient vaccine adjuvants, their mechanism of action, and factors related to low responders.
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http://dx.doi.org/10.1038/s41541-018-0078-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170373PMC
October 2018

Down selecting adjuvanted vaccine formulations: a comparative method for harmonized evaluation.

BMC Immunol 2018 01 31;19(1). Epub 2018 Jan 31.

Biomedical Primate Research Centre, Department of Parasitology, Rijswijk, The Netherlands.

Background: The need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21.

Results: The nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined.

Conclusions: The reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies.
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http://dx.doi.org/10.1186/s12865-018-0245-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793412PMC
January 2018

The anti-influenza M2e antibody response is promoted by XCR1 targeting in pig skin.

Sci Rep 2017 08 9;7(1):7639. Epub 2017 Aug 9.

VIM-INRA-Université Paris-Saclay, Domaine de Vilvert, 78350, Jouy-en-Josas, France.

XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.
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http://dx.doi.org/10.1038/s41598-017-07372-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550447PMC
August 2017

Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis.

Ther Adv Vaccines 2017 Apr 3;5(2):31-38. Epub 2017 May 3.

Infectious Disease Research Institute, Department of Global Health, University of Washington, 1616 Eastlake Ave E Ste 400, Seattle, WA 98102, USA.

Objectives: Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed.

Methods: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures.

Results: The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel.

Conclusions: These methods may be useful for improving characterization approaches of antigen-adjuvant mixtures by SDS-PAGE.
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http://dx.doi.org/10.1177/2051013617702072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418890PMC
April 2017

Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid.

PLoS Negl Trop Dis 2017 01 18;11(1):e0005240. Epub 2017 Jan 18.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities.
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http://dx.doi.org/10.1371/journal.pntd.0005240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242429PMC
January 2017

QS-21 Adjuvant: Laboratory-Scale Purification Method and Formulation Into Liposomes.

Methods Mol Biol 2017 ;1494:73-86

Vaccine Formulation Laboratory, Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066, Epalinges, Switzerland.

QS-21, a saponin extracted from the tree Quillaja saponaria Molina, is a vaccine adjuvant which has been shown to elicit robust antibody and cell-mediated immune responses in a variety of preclinical and clinical studies [1]. Its purification from the natural source is a lengthy and difficult process. The commercially available saponin mixture Quil-A is a fraction of the bark extract containing a variety of saponins, including QS-21. In order to facilitate access to QS-21 at laboratory-scale amounts, we propose here a method of purification of QS-21 starting from Quil-A. In addition, we describe a protocol to appropriately formulate QS-21 into cholesterol-containing, neutral liposomes which are known to decrease QS-21's hemolytic activity while retaining the adjuvant effect. Methods for the physicochemical characterization of purified QS-21 and of the QS-21/liposome formulations are also described.
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http://dx.doi.org/10.1007/978-1-4939-6445-1_5DOI Listing
January 2018

Ag85A DNA Vaccine Delivery by Nanoparticles: Influence of the Formulation Characteristics on Immune Responses.

Vaccines (Basel) 2016 Sep 12;4(3). Epub 2016 Sep 12.

School of Pharmaceutical Sciences, University of Geneva-University of Lausanne, Rue Michel Servet 1, 1211 Geneva, Switzerland.

The influence of DNA vaccine formulations on immune responses in combination with adjuvants was investigated with the aim to increase cell-mediated immunity against plasmid DNA (pDNA) encoding Mycobacterium tuberculosis antigen 85A. Different ratios of pDNA with cationic trimethyl chitosan (TMC) nanoparticles were characterized for their morphology and physicochemical characteristics (size, zeta potential, loading efficiency and pDNA release profile) applied in vitro for cellular uptake studies and in vivo, to determine the dose-dependent effects of pDNA on immune responses. A selected pDNA/TMC nanoparticle formulation was optimized by the incorporation of muramyl dipeptide (MDP) as an immunostimulatory agent. Cellular uptake investigations in vitro showed saturation to a maximum level upon the increase in the pDNA/TMC nanoparticle ratio, correlating with increasing Th1-related antibody responses up to a definite pDNA dose applied. Moreover, TMC nanoparticles induced clear polarization towards a Th1 response, indicated by IgG2c/IgG1 ratios above unity and enhanced numbers of antigen-specific IFN-γ producing T-cells in the spleen. Remarkably, the incorporation of MDP in TMC nanoparticles provoked a significant additional increase in T-cell-mediated responses induced by pDNA. In conclusion, pDNA-loaded TMC nanoparticles are capable of provoking strong Th1-type cellular and humoral immune responses, with the potential to be further optimized by the incorporation of MDP.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041026PMC
http://dx.doi.org/10.3390/vaccines4030032DOI Listing
September 2016

Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: A comparative study of cationic liposomes and PLGA nanoparticles.

J Control Release 2016 Mar 11;226:98-106. Epub 2016 Feb 11.

Division of Drug Delivery Technology, Leiden Academic Centre for Drug Research, Leiden, The Netherlands. Electronic address:

Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.
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http://dx.doi.org/10.1016/j.jconrel.2016.02.018DOI Listing
March 2016

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Sci Rep 2016 Jan 13;6:19300. Epub 2016 Jan 13.

Vector Molecular Biology Section, LMVR, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.
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http://dx.doi.org/10.1038/srep19300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725370PMC
January 2016

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release.

Nanomaterials (Basel) 2015 Dec 17;5(4):2317-2334. Epub 2015 Dec 17.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest Ansermet 30, 1211 Geneva, Switzerland.

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.
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http://dx.doi.org/10.3390/nano5042317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304807PMC
December 2015

TRANSVAC research infrastructure - Results and lessons learned from the European network of vaccine research and development.

Vaccine 2015 Oct 8;33(41):5481-5487. Epub 2015 Feb 8.

European Vaccine Initiative, UniversitätsKlinikum Heidelberg, Im Neuenheimer Feld 326 - 3.OG, 69120, Heidelberg, Germany. Electronic address:

TRANSVAC was a collaborative infrastructure project aimed at enhancing European translational vaccine research and training. The objective of this four year project (2009-2013), funded under the European Commission's (EC) seventh framework programme (FP7), was to support European collaboration in the vaccine field, principally through the provision of transnational access (TNA) to critical vaccine research and development (R&D) infrastructures, as well as by improving and harmonising the services provided by these infrastructures through joint research activities (JRA). The project successfully provided all available services to advance 29 projects and, through engaging all vaccine stakeholders, successfully laid down the blueprint for the implementation of a permanent research infrastructure for early vaccine R&D in Europe.
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http://dx.doi.org/10.1016/j.vaccine.2015.01.079DOI Listing
October 2015

Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.

J Infect Dis 2014 May 22;209(9):1479-84. Epub 2013 Nov 22.

Adjuvant Research Group.

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.
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http://dx.doi.org/10.1093/infdis/jit636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813749PMC
May 2014

Modern Vaccines/Adjuvants Formulation--Session 2 (Plenary II): May 15-17, 2013--Lausanne, Switzerland.

Authors:
Nicolas Collin

Hum Vaccin Immunother 2013 Sep 20;9(9):2015-6. Epub 2013 Aug 20.

Vaccine Formulation Laboratory; University of Lausanne, Switzerland; Epalinges, Vaud Switzerland.

On the 15-17th May 2013, the Fourth International Conference on Modern Vaccines/Adjuvants Formulation was organized in Lausanne, Switzerland, and gathered stakeholders from academics and from the industry to discuss several challenges, advances and promises in the field of vaccine adjuvants. Plenary session 2 of the meeting was composed of four different presentations covering: (1) the recent set-up of an adjuvant technology transfer and training platform in Switzerland, (2) the proposition to revisit existing paradigms of modern vaccinology, (3) the properties of polyethyleneimine as potential new vaccine adjuvant, and (4) the progresses in the design of HIV vaccine candidates able to induce broadly neutralizing antibodies.
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http://dx.doi.org/10.4161/hv.26208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906372PMC
September 2013

Antigen sparing with adjuvanted inactivated polio vaccine based on Sabin strains.

Vaccine 2013 Feb 9;31(9):1298-304. Epub 2013 Jan 9.

Institute for Translational Vaccinology, Antonie van Leeuwenhoeklaan 9, 3720 AL Bilthoven, The Netherlands.

Six different adjuvants, each in combination with inactivated polio vaccine (IPV) produced with attenuated Sabin strains (sIPV), were evaluated for their ability to enhance virus neutralizing antibody titres (VNTs) in the rat potency model. The increase of VNTs was on average 3-, 15-, 24-fold with adjuvants after one immunization (serotypes 1, 2, and 3, respectively). Also after a boost immunization the VNTs of adjuvanted sIPV were on average another 7-20-27 times higher than after two inoculations of sIPV without adjuvant. The results indicate that it is feasible to increase the potency of inactivated polio vaccines by using adjuvants.
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http://dx.doi.org/10.1016/j.vaccine.2012.12.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570672PMC
February 2013

Technology transfer of an oil-in-water vaccine-adjuvant for strengthening pandemic influenza preparedness in Indonesia.

Vaccine 2013 Mar 8;31(12):1641-5. Epub 2012 Aug 8.

Vaccine Formulation Laboratory, Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, Epalinges CH-1066, Switzerland.

With the current enzootic circulation of highly pathogenic avian influenza viruses, the ability to increase global pandemic influenza vaccine production capacity is of paramount importance. This has been highlighted by, and is one of the main pillars of, the WHO Global Action Plan for Influenza Vaccines (GAP). Such capacity expansion is especially relevant in developing countries. The Vaccine Formulation Laboratory at University of Lausanne is engaged in the technology transfer of an antigen-sparing oil-in-water adjuvant in order to empower developing countries vaccine manufacturers to increase pandemic influenza vaccine capacity. In a one-year project funded by United States Department of Health and Human Services, the Vaccine Formulation Laboratory transferred the process know-how and associated equipment for the pilot-scale manufacturing of an oil-in-water adjuvant to Bio Farma, Indonesia's state-owned vaccine manufacturer, for subsequent formulation with H5N1 pandemic influenza vaccines. This paper describes the experience acquired and lessons learnt from this technology transfer project.
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http://dx.doi.org/10.1016/j.vaccine.2012.07.074DOI Listing
March 2013

Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo.

Arterioscler Thromb Vasc Biol 2012 Sep 12;32(9):2185-98. Epub 2012 Jul 12.

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20892, USA.

Objective: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades.

Methods And Results: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme.

Conclusions: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.
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http://dx.doi.org/10.1161/ATVBAHA.112.253906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421056PMC
September 2012