Glucopyranosyl Lipid Adjuvant-stable Emulsion Research Articles

Efficacy of an Experimental Gonococcal Lipooligosaccharide Mimitope Vaccine Requires Terminal Complement.

A safe and effective vaccine against multidrug-resistant gonorrhea is urgently needed. An experimental peptide vaccine called TMCP2 that mimics an oligosaccharide epitope in gonococcal lipooligosaccharide, when adjuvanted with glucopyranosyl lipid adjuvant-stable emulsion, elicits bactericidal immunoglobulin G and hastens clearance of gonococci in the mouse vaginal colonization model. In this study, we show that efficacy of TMCP2 requires an intact terminal complement pathway, evidenced by loss of activity in C9-/- mice or when C7 function was blocked. In conclusion, TMCP2 vaccine efficacy in the mouse vagina requires membrane attack complex. Serum bactericidal activity may serve as a correlate of protection for TMCP2.

Maintaining immunogenicity of blood stage and sexual stage subunit malaria vaccines when formulated in combination.

Eradication of Plasmodium falciparum malaria will likely require a multivalent vaccine, but the development of a highly efficacious subunit-based formulation has been challenging. We previously showed that production and immunogenicity of two leading vaccine targets, PfMSP119 (blood-stage) and Pfs25 (sexual stage), could be enhanced upon genetic fusion to merozoite surface protein 8 (PfMSP8). Here, we sought to optimize a Pfs25-based formulation for use in combination with rPfMSP1/8 with the goal of maintaining the immunogenicity of each subunit. Comparative mouse studies were conducted to assess the effects of adjuvant selection (Alhydrogel vs. glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE)) and antigen dose (2.5 vs. 0.5 μg) on the induction of anti-Pfs25 immune responses. The antibody response (magnitude, IgG subclass profile, and transmission-reducing activity (TRA)) and cellular responses (proliferation, cytokine production) generated in response to each formulation were assessed. Similarly, immunogenicity of a bivalent vaccine containing rPfMSP1/8 and rPfs25/8 was evaluated. Alum-based formulations elicited strong and comparable humoral and cellular responses regardless of antigen form (unfused rPfs25 or chimeric rPfs25/8) or dose. In contrast, GLA-SE based formulations elicited differential responses as a function of both parameters, with 2.5 μg of rPfs25/8 inducing the highest titers of functional anti-Pfs25 antibodies. Based on these data, chimeric rPfs25/8 was selected and tested in a bivalent formulation with rPfMSP1/8. Strong antibody titers against Pfs25 and PfMSP119 domains were induced with GLA-SE based formulations, with no indication of antigenic competition. We were able to generate an immunogenic bivalent vaccine designed to target multiple parasite stages that could reduce both clinical disease and parasite transmission. The use of the same PfMSP8 carrier for two different vaccine components was effective in this bivalent formulation. As such, the incorporation of additional protective targets fused to the PfMSP8 carrier into the formulation should be feasible, further broadening the protective response.

PRIMVAC vaccine adjuvanted with Alhydrogel or GLA-SE to prevent placental malaria: a first-in-human, randomised, double-blind, placebo-controlled study

PRIMVAC is a VAR2CSA-derived placental malaria vaccine candidate aiming to prevent serious clinical outcomes of Plasmodium falciparum infection during pregnancy. We assessed the safety and immunogenicity of PRIMVAC adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) in French and Burkinabe women who were not pregnant. This first-in-human, randomised, double-blind, placebo-controlled, dose escalation trial was done in two staggered phases, a phase 1A trial in 18-35-year-old women who were malaria naive in a hospital in France and a subsequent phase 1B trial in women who were naturally exposed to P falciparum and nulligravid in the clinical site of a research centre in Burkina Faso. Volunteers were recruited into four sequential cohorts receiving PRIMVAC intramuscularly at day 0, 28, and 56: two cohorts in France receiving 20 μg or 50 μg of PRIMVAC and then two in Burkina Faso receiving 50 μg or 100 μg of PRIMVAC. Volunteers were randomly assigned (1:1) to two groups (PRIMVAC adjuvanted with either Alhydrogel or GLA-SE) in France and randomly assigned (2:2:1) to three groups (PRIMVAC adjuvanted with either Alhydrogel, GLA-SE, or placebo) in Burkina Faso. Randomisation was centralised, using stratification by cohort and blocks of variable size, and syringes were masked by opaque labels. The primary endpoint was the proportion of participants with any grade 3 or higher adverse reaction to vaccination up until day 35. Safety at later time points as well as humoral and cellular immunogenicity were assessed in secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02658253. Between April 19, 2016, and July 13, 2017, 68 women (18 in France, 50 in Burkina Faso) of 101 assessed for eligibility were included. No serious adverse event related to the vaccine occurred. PRIMVAC antibody titres increased with each dose and seroconversion was observed in all women vaccinated with PRIMVAC (n=57). PRIMVAC antibody titres reached a peak (geometric mean 11 843·0, optical density [OD] 1·0, 95% CI 7559·8-18 552·9 with 100 μg dose and GLA-SE) 1 week after the third vaccination (day 63). Compared with Alhydrogel, GLA-SE tended to improve the PRIMVAC antibody response (geometric mean 2163·5, OD 1·0, 95% CI 1315·7-3557·7 with 100 μg dose and Alhydrogel at day 63). 1 year after the last vaccination, 20 (71%) of 28 women who were vaccinated with PRIMVAC/Alhydrogel and 26 (93%) of 28 women who were vaccinated with PRIMVAC/GLA-SE still had anti-PRIMVAC antibodies, although antibody magnitude was markedly lower (452·4, OD 1·0, 95% CI 321·8-636·1 with 100 μg dose and GLA-SE). These antibodies reacted with native homologous VAR2CSA expressed by NF54-CSA infected erythrocytes (fold change from baseline at day 63 with 100 μg dose and GLA-SE: 10·74, 95% CI 8·36-13·79). Limited cross-recognition, restricted to sera collected from women that received the 100 μg PRIMVAC dose, was observed against heterologous VAR2CSA variants expressed by FCR3-CSA (fold change from baseline at day 63: 1·49, 95% CI 1·19-1·88) and 7G8-CSA infected erythrocytes (1·2, 1·08-1·34). PRIMVAC adjuvanted with Alhydrogel or GLA-SE had an acceptable safety profile, was immunogenic, and induced functional antibodies reacting with the homologous VAR2CSA variant expressed by NF54-CSA infected erythrocytes. Cross-reactivity against heterologous VAR2CSA variants was limited and only observed in the higher dose group. An alternate schedule of immunisation, antigen dose, and combinations with other VAR2CSA-based vaccines are envisaged to improve the cross-reactivity against heterologous VAR2CSA variants. Bundesministerium für Bildung und Forschung, through Kreditanstalt für Wiederaufbau, Germany; Inserm, and Institut National de Transfusion Sanguine, France; Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Malaria vaccine candidate based on Duffy-binding protein elicits strain transcending functional antibodies in a Phase I trial

Reticulocyte invasion by Plasmodium vivax requires interaction of the Duffy-binding protein (PvDBP) with host Duffy antigen receptor for chemokines (DARCs). The binding domain of PvDBP maps to a cysteine-rich region referred to as region II (PvDBPII). Blocking this interaction offers a potential path to prevent P. vivax blood-stage growth and P. vivax malaria. This forms the rationale for development of a vaccine based on PvDBPII. Here we report results of a Phase I randomized trial to evaluate the safety and immunogenicity of recombinant PvDBPII formulated with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Thirty-six malaria-naive, healthy Indian male subjects aged 18–45 years were assigned into three cohorts corresponding to doses of 10, 25 and 50 µg of PvDBPII formulated with 5 µg of GLA-SE. Each cohort included nine PvDBPII/GLA-SE vaccinees and three hepatitis B control vaccine recipients. Each subject received the assigned vaccine intramuscularly on days 0, 28 and 56, and was followed up till day 180. No serious AE was reported and PvDBPII/GLA-SE was well-tolerated and safe. Analysis by ELISA showed that all three doses of PvDBPII elicited antigen-specific binding-inhibitory antibodies. The 50 µg dose elicited antibodies against PvDBPII that had the highest binding-inhibitory titres and were most persistent. Importantly, the antibody responses were strain transcending and blocked receptor binding of diverse PvDBP alleles. These results support further clinical development of PvDBPII/GLA-SE to evaluate efficacy against sporozoite or blood-stage challenge in controlled human malaria infection (CHMI) models and against natural P. vivax challenge in malaria endemic areas.

Evaluating the Vaccine Potential of a Tetravalent Fusion Protein (rBmHAXT) Vaccine Antigen Against Lymphatic Filariasis in a Mouse Model.

Lymphatic filariasis (LF) is a tropical parasitic infection of human transmitted by mosquitoes. Chronic infection results in severe physical disability in the infected patients. Although several potential vaccine antigens were identified by several groups, there are no licensed prophylactic vaccine to date against this infection in the human. Previous attempts from our laboratory to develop a trivalent prophylactic vaccine against LF showed that >90% protection could be achieved in rodent models. However, this trivalent vaccine gave only 35% protection in non-human primates. The major focus of this study was to develop a tetravalent prophylactic vaccine (rBmHAXT) and test the vaccine potential in a mouse model. We evaluated three different adjuvant formulations; alum, glucopyranosyl lipid adjuvant in stable emulsion (GLA/SE) alum (AL019), and mannosylated chitosan (MCA) to determine the optimum adjuvant formulation for rBmHAXT. Results presented in this study show that rBmHAXT + AL019 gave the highest rate of protection (>88%) against challenge infection, compared to rBmHAXT + AL007 (79%), rBmHAXT + MCA (79%) and controls. Analysis of the immune correlates of protection showed that all three adjuvants elicited high titer of antigen-specific IgG1, IgG2a, and IgG2b antibodies. High number of IFN-γ-producing antigen-specific memory cells were generated in the vaccinated animals irrespective of the adjuvants used. Similarly, spleen cells from rBmHAXT-vaccinated animals secreted IL-4, IL-10, and IFN-γ in response to rBmHAXT suggesting the generation of a balanced Th1/Th2 response. There was also an increase in IL-17-secreting cells in rBmHAXT-vaccinated animals. These findings thus suggest that rBmHAXT + AL019 is a better prophylactic formulation for LF.

Prospects of developing a prophylactic vaccine against human lymphatic filariasis – evaluation of protection in non-human primates

Lymphatic filariasis (LF) affects 120 million people around the world and another 856 million people are at risk of acquiring the infection. Mass Drug Administration (MDA) spearheaded by the World Health Organization is the only current strategy to control this infection. Recent reports suggest that despite several rounds of MDA, elimination has not been achieved and there is a need for more stringent control strategies for control of LF. An effective prophylactic vaccine combined with MDA has significant potential. Initial trials using a prophylactic trivalent recombinant Brugia malayi heat shock protein 12.6, abundant larval transcript -2 and tetraspanin large extra-cellular loop (rBmHAT) vaccine developed in our laboratory conferred only 35% protection in macaques. Therefore, the focus of the present study was to improve the current vaccine formulation to obtain better protection in non-human primates. We made two modifications to the current formulation: (i) the addition of another antigen, thioredoxin peroxidase-2 (TPX-2) to make it a tetravalent vaccine (rBmHAXT) and (ii) the inclusion of an adjuvant; AL019 (alum plus glucopyranosyl lipid adjuvant-stable emulsion) that is known to promote a balanced Th1/Th2 response. A double-blinded vaccination trial was performed with 40 macaques that were divided into three treatment groups and one control group (n = 10/group). Vaccinated animals received 4 immunisations at 1 month intervals with 150 µg/ml of rBmHAT plus alum, rBmHAT plus AL019 or rBmHAXT plus AL019. Control animals received AL019 only. All vaccinated macaques developed significant (P ≤ 0.003) titers of antigen-specific IgG antibodies (1:20,000) compared with the controls. One month after the last dose, all macaques were challenged s.c. with 130–180 B. malayi L3s. Our results showed that seven out of 10 (70%) of macaques given the improved rBmHAXT vaccine did not develop the infection compared with AL019 controls, of which seven out of 10 macaques developed the infection. Titers of antigen-specific IgG1 and IgG2 antibodies were significantly (P ≤ 0.01) higher in vaccinated animals and there was an increase in the percentage of IL-4 and IFN-γ secreting antigen-responding memory T cells. These studies demonstrated that the improved formulation (rBmHAXT plus AL019) is a promising vaccine candidate against human lymphatic filariasis.

LepVax, a defined subunit vaccine that provides effective pre-exposure and post-exposure prophylaxis of M. leprae infection

Sustained elimination of leprosy as a global health concern likely requires a vaccine. The current standard, BCG, confers only partial protection and precipitates paucibacillary (PB) disease in some instances. When injected into mice with the T helper 1 (Th1)-biasing adjuvant formulation Glucopyranosyl Lipid Adjuvant in stable emulsion (GLA-SE), a cocktail of three prioritized antigens (ML2055, ML2380 and ML2028) reduced M. leprae infection levels. Recognition and protective efficacy of a single chimeric fusion protein incorporating these antigens, LEP-F1, was confirmed in similar experiments. The impact of post-exposure immunization was then assessed in nine-banded armadillos that demonstrate a functional recapitulation of leprosy. Armadillos were infected with M. leprae 1 month before the initiation of post-exposure prophylaxis. While BCG precipitated motor nerve conduction abnormalities more rapidly and severely than observed for control infected armadillos, motor nerve injury in armadillos treated three times, at monthly intervals with LepVax was appreciably delayed. Biopsy of cutaneous nerves indicated that epidermal nerve fiber density was not significantly altered in M. leprae-infected animals although Remak Schwann cells of the cutaneous nerves in the distal leg were denser in the infected armadillos. Importantly, LepVax immunization did not exacerbate cutaneous nerve involvement due to M. leprae infection, indicating its safe use. There was no intraneural inflammation but a reduction of intra axonal edema suggested that LepVax treatment might restore some early sensory axonal function. These data indicate that post-exposure prophylaxis with LepVax not only appears safe but, unlike BCG, alleviates and delays the neurologic disruptions caused by M. leprae infection.

Humoral and cell-mediated immune responses to H5N1 plant-made virus-like particle vaccine are differentially impacted by alum and GLA-SE adjuvants in a Phase 2 clinical trial

The hemagglutinination inhibition (HI) response remains the gold standard used for the licensure of influenza vaccines. However, cell-mediated immunity (CMI) deserves more attention, especially when evaluating H5N1 influenza vaccines that tend to induce poor HI response. In this study, we measured the humoral response (HI) and CMI (flow cytometry) during a Phase II dose-ranging clinical trial (NCT01991561). Subjects received two intramuscular doses, 21 days apart, of plant-derived virus-like particles (VLP) presenting the A/Indonesia/05/2005 H5N1 influenza hemagglutinin protein (H5) at the surface of the VLP (H5VLP). The vaccine was co-administrated with Alhydrogel® or with a glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). We demonstrated that low doses (3.75 or 7.5 μg H5VLP) of GLA-SE-adjuvanted vaccines induced HI responses that met criteria for licensure at both antigen doses tested. Alhydrogel adjuvanted vaccines induced readily detectable HI response that however failed to meet licensure criteria at any of three doses (10, 15 and 20 μg) tested. The H5VLP also induced a sustained (up to 6 months) polyfunctional and cross-reactive HA-specific CD4+ T cell response in all vaccinated groups. Interestingly, the frequency of central memory Th1-primed precursor cells before the boost significantly correlated with HI titers 21 days after the boost. The ability of the low dose GLA-SE-adjuvanted H5VLP to elicit both humoral response and a sustained cross-reactive CMI in healthy adults is very attractive and could result in significant dose-sparing in a pandemic situation.

The Plasmodium falciparum Cell-Traversal Protein for Ookinetes and Sporozoites as a Candidate for Preerythrocytic and Transmission-Blocking Vaccines.

ABSTRACTRecent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo. Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.

Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer.

BackgroundBuruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters.Methodology/Principal FindingsTo assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a TH1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model.ConclusionsEven though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci.

Production and preclinical evaluation of Plasmodium falciparum MSP-119 and MSP-311 chimeric protein, PfMSP-Fu24.

A Plasmodium falciparum chimeric protein, PfMSP-Fu24, was constructed by genetically coupling immunodominant, conserved regions of two merozoite surface proteins, the 19-kDa region C-terminal region of merozoite surface protein 1 (PfMSP-119) and an 11-kDa conserved region of merozoite surface protein 3 (PfMSP-311), to augment the immunogenicity potential of these blood-stage malaria vaccine candidates. Here we describe an improved, efficient, and scalable process to produce high-quality PfMSP-Fu24. The chimeric protein was produced in Escherichia coli SHuffle T7 Express lysY cells that express disulfide isomerase DsbC. A two-step purification process comprising metal affinity followed by cation exchange chromatography was developed, and we were able to obtain PfMSP-Fu24 with purity above 99% and with a considerable yield of 23 mg/liter. Immunogenicity of PfMSP-Fu24 formulated with several adjuvants, including Adjuplex, Alhydrogel, Adjuphos, Alhydrogel plus glucopyranosyl lipid adjuvant, aqueous (GLA-AF), Adjuphos+GLA-AF, glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), and Freund's adjuvant, was evaluated. PfMSP-Fu24 formulated with GLA-SE and Freund's adjuvant in mice and with Alhydrogel and Freund's adjuvant in rabbits produced high titers of PfMSP-119 and PfMSP-311-specific functional antibodies. Some of the adjuvant formulations induced inhibitory antibody responses and inhibited in vitro growth of P. falciparum parasites in the presence as well as in the absence of human monocytes. These results suggest that PfMSP-Fu24 can form a constituent of a multistage malaria vaccine.

Cross-species protection: Schistosoma mansoni Sm-p80 vaccine confers protection against Schistosoma haematobium in hamsters and baboons

The ability of the Schistosoma mansoni antigen, Sm-p80, to provide cross-species protection against Schistosoma haematobium challenge was evaluated in hamster and baboon models. Pronounced reduction in worm burden (48%) and in tissue egg load (64%) was observed in hamsters vaccinated with recombinant Sm-p80 admixed with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Similarly, in baboons, the Sm-p80/GLA-SE vaccine produced a 25% reduction in S. haematobium adult worms and decreased the egg load in the urinary bladder by 64%. A 40% and 53% reduction in fecal and urine egg output, respectively, was observed in vaccinated baboons. A balanced pro-inflammatory (Th17 and Th1) and Th2 type of response was generated after vaccination and appears indicative of augmented prophylactic efficacy. These data on cross-species protection coupled with the prophylactic, therapeutic and antifecundity efficacy against the homologous parasite, S. mansoni, reinforces Sm-p80 as a promising vaccine candidate. It is currently being prepared for GMP-compliant manufacture and for further pre-clinical development leading to human clinical trials. These results solidify the expectation that the Sm-p80 vaccine will provide relief for both the intestinal and the urinary schistosomiasis and thus will be greatly beneficial in reducing the overall burden of schistosomiasis.

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly-functional T(H)1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR-domain-containing adapter inducing IFN-beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T(H)1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA-SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild-type mice. We find that both MyD88 and TRIF are necessary for GLA-SE to induce a poly-functional T(H)1 immune response characterized by CD4(+) T cells producing IFN-γ, TNF, and IL-2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA-SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T(H)1-skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.

Full-length Plasmodium falciparum circumsporozoite protein administered with long-chain poly(I·C) or the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion elicits potent antibody and CD4+ T cell immunity and protection in mice.

The Plasmodium falciparum circumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-length P. falciparum CS proteins expressed in Escherichia coli or Pichia pastoris for their ability to induce immunity and protection in mice when administered with long-chain poly(I · C) [poly(I · C)LC] as an adjuvant. CS proteins expressed in E. coli induced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4(+) T cell responses. Moreover, E. coli-derived CS proteins in combination with poly(I · C)LC induced potent multifunctional (interleukin 2-positive [IL-2(+)], tumor necrosis factor alpha-positive [TNF-α(+)], gamma interferon-positive [IFN-γ(+)]) CD4(+) effector T cell responses in blood, in spleen, and particularly in liver. Using transgenic Plasmodium berghei expressing the repeat region of P. falciparum CSP [Pb-CS(Pf)], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ~50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4(+) T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4(+) T cell immunity that was significantly less potent than that with poly(I · C)LC. Overall, these data suggest that full-length CS proteins and poly(I · C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.

Novel Structurally Designed Vaccine for S. aureus α-Hemolysin: Protection against Bacteremia and Pneumonia

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.

GLA-SE, a Synthetic Toll-like Receptor 4 Agonist, Enhances T-Cell Responses to Influenza Vaccine in Older Adults

The decline in influenza vaccine efficacy in older adults is associated with a limited ability of current split-virus vaccines (SVVs) to stimulate cytotoxic T lymphocyte (CTL) responses required for clinical protection against influenza. The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) was combined with SVV to stimulate peripheral blood mononuclear cells (PBMCs) in vitro to determine the cytokine response in dendritic cell subsets. Stimulated PBMCs were then challenged with live influenza virus to mimic the response to natural infection following vaccination, using previously identified T-cell correlates of protection. GLA-SE significantly increased the proportion of myeloid dendritic cells that produced tumor necrosis factor α, interleukin 6, and interleukin 12. When combined with SVV to stimulate PBMCs in vitro, this effect of GLA-SE was shown to regulate a T-helper 1 cell response upon challenge with live influenza virus; interleukin 10 production was suppressed, thus significantly increasing the interferon γ to interleukin 10 ratio and the cytolytic (granzyme B) response to influenza virus challenge, both of which have been shown to correlate with protection against influenza in older adults. Our findings suggest that a novel adjuvant, GLA-SE, combined with standard SVV has the potential to significantly improve vaccine-mediated protection against influenza in older adults.

A Synthetic Adjuvant to Enhance and Expand Immune Responses to Influenza Vaccines

Safe, effective adjuvants that enhance vaccine potency, including induction of neutralizing Abs against a broad range of variant strains, is an important strategy for the development of seasonal influenza vaccines which can provide optimal protection, even during seasons when available vaccines are not well matched to circulating viruses. We investigated the safety and ability of Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE), a synthetic Toll-like receptor (TLR)4 agonist formulation, to adjuvant Fluzone® in mice and non-human primates. The GLA-SE adjuvanted Fluzone vaccine caused no adverse reactions, increased the induction of T helper type 1 (TH1)-biased cytokines such as IFNγ, TNF and IL-2, and broadened serological responses against drifted A/H1N1 and A/H3N2 influenza variants. These results suggest that synthetic TLR4 adjuvants can enhance the magnitude and quality of protective immunity induced by influenza vaccines.