Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice.

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 cost. 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.

Activation of CD4 T cells during prime immunization determines the success of a therapeutic hepatitis B vaccine in HBV-carrier mouse models.

BACKGROUND & AIMS: We recently developed a heterologous therapeutic vaccination scheme (TherVacB) comprising a particulate protein prime followed by a modified vaccinia-virus Ankara (MVA)-vector boost for the treatment of HBV. However, the key determinants required to overcome HBV-specific immune tolerance remain unclear. Herein, we aimed to study new combination adjuvants and unravel factors that are essential for the antiviral efficacy of TherVacB. METHODS: Recombinant hepatitis B surface and core antigen (HBsAg and HBcAg) particles were formulated with different liposome- or oil-in-water emulsion-based combination adjuvants containing saponin QS21 and monophosphoryl lipid A; these formulations were compared to STING-agonist c-di-AMP and conventional aluminium hydroxide formulations. Immunogenicity and the antiviral effects of protein antigen formulations and the MVA-vector boost within TherVacB were evaluated in adeno-associated virus-HBV-infected and HBV-transgenic mice. RESULTS: Combination adjuvant formulations preserved HBsAg and HBcAg integrity for ≥12 weeks, promoted human and mouse dendritic cell activation and, within TherVacB, elicited robust HBV-specific antibody and T-cell responses in wild-type and HBV-carrier mice. Combination adjuvants that prime a balanced HBV-specific type 1 and 2 T helper response induced high-titer anti-HBs antibodies, cytotoxic T-cell responses and long-term control of HBV. In the absence of an MVA-vector boost or following selective CD8 T-cell depletion, HBsAg still declined (mediated mainly by anti-HBs antibodies) but HBV replication was not controlled. Selective CD4 T-cell depletion during the priming phase of TherVacB resulted in a complete loss of vaccine-induced immune responses and its therapeutic antiviral effect in mice. CONCLUSIONS: Our results identify CD4 T-cell activation during the priming phase of TherVacB as a key determinant of HBV-specific antibody and CD8 T-cell responses. IMPACT AND IMPLICATIONS: Therapeutic vaccination is a potentially curative treatment option for chronic hepatitis B. However, it remains unclear which factors are essential for breaking immune tolerance in HBV carriers and determining successful outcomes. Our study provides the first direct evidence that efficient priming of HBV-specific CD4 T cells determines the success of therapeutic hepatitis B vaccination in two preclinical HBV-carrier mouse models. Applying an optimal formulation of HBV antigens that activates CD4 and CD8 T cells during prime immunization provided the foundation for an antiviral effect of therapeutic vaccination, while depletion of CD4 T cells led to a complete loss of vaccine-induced antiviral efficacy. Boosting CD8 T cells was important to finally control HBV in these mouse models. Our findings provide important insights into the rational design of therapeutic vaccines for the cure of chronic hepatitis B.

Meeting vaccine formulation challenges in an emergency setting: Towards the development of accessible vaccines.

Vaccination is considered one of the most successful strategies to prevent infectious diseases. In the event of a pandemic or epidemic, the rapid development and distribution of the vaccine to the population is essential to reduce mortality, morbidity and transmission. As seen during the COVID-19 pandemic, the production and distribution of vaccines has been challenging, in particular for resource-constrained settings, essentially slowing down the process of achieving global coverage. Pricing, storage, transportation and delivery requirements of several vaccines developed in high-income countries resulted in limited access for low-and-middle income countries (LMICs). The capacity to manufacture vaccines locally would greatly improve global vaccine access. In particular, for the development of classical subunit vaccines, the access to vaccine adjuvants is a pre-requisite for more equitable access to vaccines. Vaccine adjuvants are agents required to augment or potentiate, and possibly target the specific immune response to such type of vaccine antigens. Openly accessible or locally produced vaccine adjuvants may allow for faster immunization of the global population. For local research and development of adjuvanted vaccines to expand, knowledge on vaccine formulation is of paramount importance. In this review, we aim to discuss the optimal characteristics of a vaccine developed in an emergency setting by focusing on the importance of vaccine formulation, appropriate use of adjuvants and how this may help overcome barriers for vaccine development and production in LMICs, achieve improved vaccine regimens, delivery and storage requirements.

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

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 (RDSD28-D56 -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.

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

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.

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

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.

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

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.

Efficacy of a stable broadly protective subunit vaccine platform against SARS-CoV-2 variants of concern.

The emergence and ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid vaccine development platforms that can be updated to counteract emerging variants of currently circulating and future emerging coronaviruses. Here we report the development of a "train model" subunit vaccine platform that contains a SARS-CoV-2 Wuhan S1 protein (the "engine") linked to a series of flexible receptor binding domains (RBDs; the "cars") derived from SARS-CoV-2 variants of concern (VOCs). We demonstrate that these linked subunit vaccines when combined with Sepivac SWE™, a squalene in water emulsion (SWE) adjuvant, are immunogenic in Syrian hamsters and subsequently provide protection from infection with SARS-CoV-2 VOCs Omicron (BA.1), Delta, and Beta. Importantly, the bivalent and trivalent vaccine candidates offered protection against some heterologous SARS-CoV-2 VOCs that were not included in the vaccine design, demonstrating the potential for broad protection against a range of different VOCs. Furthermore, these formulated vaccine candidates were stable at 2-8 °C for up to 13 months post-formulation, highlighting their utility in low-resource settings. Indeed, our vaccine platform will enable the development of safe and broadly protective vaccines against emerging betacoronaviruses that pose a significant health risk for humans and agricultural animals.

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.

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.

Glycosylated nanoparticle-based PfCSP vaccine confers long-lasting antibody responses and sterile protection in mouse malaria model.

The development of an effective and durable vaccine remains a central goal in the fight against malaria. Circumsporozoite protein (CSP) is the major surface protein of sporozoites and the target of the only licensed Plasmodium falciparum (Pf) malaria vaccine, RTS,S/AS01. However, vaccine efficacy is low and short-lived, highlighting the need for a second-generation vaccine with superior efficacy and durability. Here, we report a Helicobacter pylori apoferritin-based nanoparticle immunogen that elicits strong B cell responses against PfCSP epitopes that are targeted by the most potent human monoclonal antibodies. Glycan engineering of the scaffold and fusion of an exogenous T cell epitope enhanced the anti-PfCSP B cell response eliciting strong, long-lived and protective humoral immunity in mice. Our study highlights the power of rational vaccine design to generate a highly efficacious second-generation anti-infective malaria vaccine candidate and provides the basis for its further development.

OVX033, a nucleocapsid-based vaccine candidate, provides broad-spectrum protection against SARS-CoV-2 variants in a hamster challenge model.

Spike-based COVID-19 vaccines induce potent neutralizing antibodies but their efficacy against SARS-CoV-2 variants decreases. OVX033 is a recombinant protein composed of the full-length nucleocapsid (N) protein of SARS-CoV-2 genetically fused to oligoDOM®, a self-assembling domain which improves antigen immunogenicity. OVX033 including N as an antigenic target is proposed as new vaccine candidate providing broad-spectrum protection against sarbecoviruses. OVX033 demonstrated its ability to trigger cross-reactive T cell responses and cross-protection against three variants of SARS-CoV-2 (B.1 Europe, Delta B.1.617.2, and Omicron B.1.1.529) in a hamster challenge model, as evidenced by lower weight loss, lower lung viral loads, and reduced lung histopathological lesions.

Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms.

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.

Potential business model for a European vaccine R&D infrastructure and its estimated socio-economic impact.

Background: Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods: In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results: Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions: The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.

Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed ß-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

Designing Adjuvant Formulations to Promote Immunogenicity and Protective Efficacy of Leptospira Immunoglobulin-Like Protein A Subunit Vaccine.

The leptospirosis burden on humans, especially in high-risk occupational groups and livestock, leads to public health and economic problems. Leptospirosis subunit vaccines have been under development and require further improvement to provide complete protection. Adjuvants can be used to enhance the amplitude, quality, and durability of immune responses. Previously, we demonstrated that LMQ adjuvant (neutral liposomes containing monophosphoryl lipid A (MPL) and Quillaja saponaria derived QS21 saponin) promoted protective efficacy of LigAc vaccine against Leptospira challenge. To promote immunogenicity and protective efficacy of the subunit vaccines, three alternative adjuvants based on neutral liposomes or squalene-in-water emulsion were evaluated in this study. LQ and LQuil adjuvants combined the neutral liposomes with the QS21 saponin or Quillaja saponaria derived QuilA® saponin, respectively. SQuil adjuvant combined a squalene-in-water emulsion with the QuilA® saponin. The immunogenicity and protective efficacy of LigAc (20 µg) formulated with the candidate adjuvants were conducted in golden Syrian hamsters. Hamsters were vaccinated three times at a 2-week interval, followed by a homologous challenge of L. interrogans serovar Pomona. The results showed that LigAc combined with LQ, LQuil, or SQuil adjuvants conferred substantial antibody responses and protective efficacy (survival rate, pathological change, and Leptospira renal colonization) comparable to LMQ adjuvant. The LigAc+LQ formulation conferred 62.5% survival but was not significantly different from LigAc+LMQ, LigAc+LQuil, and LigAc+SQuil formulations (50% survival). This study highlights the potential of saponin-containing adjuvants LMQ, LQ, LQuil, and SQuil for both human and animal leptospirosis vaccines.

Characterization and comparison of novel adjuvants for a prefusion clamped MERS vaccine.

Various chemical adjuvants are available to augment immune responses to non-replicative, subunit vaccines. Optimized adjuvant selection can ensure that vaccine-induced immune responses protect against the diversity of pathogen-associated infection routes, mechanisms of infectious spread, and pathways of immune evasion. In this study, we compare the immune response of mice to a subunit vaccine of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) spike protein, stabilized in its prefusion conformation by a proprietary molecular clamp (MERS SClamp) alone or formulated with one of six adjuvants: either (i) aluminium hydroxide, (ii) SWE, a squalene-in-water emulsion, (iii) SQ, a squalene-in-water emulsion containing QS21 saponin, (iv) SMQ, a squalene-in-water emulsion containing QS21 and a synthetic toll-like receptor 4 (TLR4) agonist 3D-6-acyl Phosphorylated HexaAcyl Disaccharide (3D6AP); (v) LQ, neutral liposomes containing cholesterol, 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and QS21, (vi) or LMQ, neutral liposomes containing cholesterol, DOPC, QS21, and 3D6AP. All adjuvanted formulations induced elevated antibody titers which where greatest for QS21-containing formulations. These had elevated neutralization capacity and induced higher frequencies of IFNƔ and IL-2-producing CD4+ and CD8+ T cells. Additionally, LMQ-containing formulations skewed the antibody response towards IgG2b/c isotypes, allowing for antibody-dependent cellular cytotoxicity. This study highlights the utility of side-by-side adjuvant comparisons in vaccine development.

Sand fly salivary proteins induce strong cellular immunity in a natural reservoir of visceral leishmaniasis with adverse consequences for Leishmania.

Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-gamma at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG(2) antibody levels and significant IFN-gamma production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-gamma and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response.

Plasmodium falciparum: nitric oxide modulates heme speciation in isolated food vacuoles.

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b(5), localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b(5) reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.

Better Pandemic Influenza Preparedness through Adjuvant Technology Transfer: Challenges and Lessons Learned.

Adequate global vaccine coverage during an influenza pandemic is essential to mitigate morbidity, mortality, and economic impact. Vaccine development and production needs to be sufficient to meet a vast global demand, requiring international cooperation and local vaccine production capacity, especially in resource-constrained countries. The use of adjuvants is one approach to augment the number of available vaccine doses and to overcome potential vaccine shortages. Appropriately selected adjuvant technologies can decrease the amount of vaccine antigen required per dose, may broaden or lengthen the conferred protection against disease, and may even allow protective single-dose vaccination. Here we describe a technology transfer collaboration between Switzerland and Indonesia that led to the establishment of a vaccine formulation platform in Surabaya which involved the transfer of equipment and expertise to enable research and development of adjuvanted vaccine formulations and delivery systems. This new Indonesian capability aims to facilitate local and regional access to know-how relating to adjuvanted vaccine formulations, thus promoting their application to local vaccine developers. In this review, we aim to share the "lessons learned" from this project to both support and inspire future scientific collaborations of a similar nature.

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

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.