Microparticles in MF59, a potent adjuvant combination for a recombinant protein vaccine against HIV-1

Novel adjuvant formulations involving PLG microparticles with entrapped recombinant protein antigens (env gp120 and p24 gag) from human immunodeficiency virus type-1 (HIV-1), dispersed in the emulsion adjuvant MF59 were evaluated as potential HIV-1 vaccine candidates in mice and baboons. In mice, the adjuvant combination induced significantly enhanced antibody responses in comparison to either adjuvant used alone. In addition, the polylactide co-glycolide polymer (PLG) microparticles and MF59 combination induced CTL activity against HIV-1 p24 gag. In baboons, the adjuvant combination induced significantly enhanced antibody titers after a single dose of gp120, but the responses were comparable to gp120 in MF59 alone after boosting. Both MF59+gp120 alone and PLG/gp120 in MF59 induced neutralizing antibodies against a T cell line-adapted (TCLA) strain and a primary isolate of HIV-1. In contrast to the observations with gp120, immunization in baboons with PLG/p24 in MF59 induced significantly enhanced antibody responses after boosting, in comparison to immunization with MF59 alone + p24.     

Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice

Influenza is controlled by protective titres of neutralizing antibodies, induced with the help of CD4 T-cells, and by antiviral T-cell effector function. Adjuvants are essential for the efficient vaccination of a na?ve population against avian influenza. We evaluated a range of adjuvants for their ability to enhance, in na?ve mice, protective hemagglutination inhibition (HI) titres, which represent the generally accepted correlate of protection, virus-neutralizing titres and T-cell responses to a new generation influenza vaccine produced in cell culture. The selected adjuvants include alum, calcium phosphate (CAP), MF59, the delivery system poly-(lactide co-glycolide) (PLG) and the immune potentiator CpG. MF59 was clearly the most potent single adjuvant and induced significantly enhanced, long-lasting HI and neutralizing titres and T-cell responses in comparison to all alternatives. The combination of alum, MF59, CAP or PLG with CpG generally induced slightly more potent titres. The addition of CpG to MF59 also induced a more potent Th1 cellular immune response, represented by higher IgG2a titres and the induction of a strongly enhanced IFN-gamma response in splenocytes from immunized mice. These observations have significant implications for the development of new and improved flu vaccines against pandemic and inter-pandemic influenza virus strains.     

Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid

The use of biodegradable polymer matrices as a single-dose vaccine delivery system was investigated using tetanus toxoid (TT) and diphtheria toxoid (DT). BALB/c mice were immunized with TT or DT in different formulations including individual, in minipellet and aluminum hydroxide (alum), and the antibody responses were monitored for 48 weeks. Antigens entrapped in minipellet elicited higher antibody responses compared to those obtained with individual antigens and antigens adsorbed to alum and the antibody levels remained elevated over 48 weeks. In addition, minipellet formulations induced the same subclasses of antibodies induced by alum formulations. These results raise the possibility to obtain optimal and long-lasting immune responses by a single administration of minipellet formulations.     

Development of hepatitis B oral vaccine using B-cell epitope loaded PLG microparticles

Oral hepatitis B vaccine formulation was prepared by successful encapsulation of immunogenic peptide representing residues 127-145 of the immunodominant B-cell epitope of hepatitis B surface antigen (HBsAg) in poly(D,L-lactide co-glycolide) (PLG) microparticles. The smooth, spherical PLG microparticles with a diameter of around 10 microm was prepared by using W/O/W double emulsion solvent evaporation method. The entrapment efficiency of B-cell epitope peptide (BCEP) into PLG microparticles was 64%. In vitro studies showed B-cell epitope loaded PLG microparticles (BCEM) released the peptide in sustained profile and reached 64.9% efficiency by Day 25. Single oral immunization of mice with BCEM led to the significant induction of specific serum IgG and IgM anti-HB antibodies. After the termination of antibody induction, the orally immunized mice were infected with HBsAg, which resulted in the rapid production of antibodies against HBsAg as a result of secondary immune response. PLG microparticles formulation approach may have potential in increasing the efficacy of microparticulate systems for the oral administration of hepatitis B vaccine.     

Analysis of the immunogenicity and bioactivities of a split influenza A/H7N9 vaccine mixed with MF59 adjuvant in BALB/c mice

The H7N9 influenza virus caused significant mortality and morbidity in humans during an outbreak in China in 2013. A recombinant H7N9 influenza seed with hemagglutinin (HA) and neuraminidase (NA) gene segments from A/Zhejiang/DTID-ZJU01/2013(H7N9) and six internal protein gene segments from A/Puerto Rico/8/34(H1N1; PR8) were generated using reverse genetics. We sought to determine the immunogenic, protective properties, and mechanisms of a split avian influenza A/H7N9 vaccine mixed with MF59 adjuvant in comparison to vaccines that included other adjuvant. BALB/c mice were vaccinated with two doses of different amounts and combinations of this novel A/ZJU01/PR8/2013 split vaccine with adjuvant. Mice were subsequently challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) by intranasal inoculation. We verified that MF59 enhanced the HI, MN, and IgG antibody titers to influenza antigens. Compared with alum, MF59 could more potentially induce humoral immune responses and Th2 cytokine production after virus infection, while both MF59 and alum can slightly increase NK cell activity. This split H7N9 influenza vaccine with MF59 adjuvant could effectively induce antibody production and protect mice from H7N9 virus challenge. We have selected this vaccine for manufacture and future clinical studies to protect humans from H7N9 virus infection.     

Preparation and characterization of an immunogenic meningococcal group A conjugate vaccine for use in Africa

Periodic epidemics of group A meningococcal (Mn A) meningitis continue to occur in sub-Saharan Africa. For its prevention, a Mn A polysaccharide (PS)–tetanus toxoid (TT) conjugate vaccine was developed using reductive amination of polysaccharide aldehydes and toxoid hydrazides. In mouse immunization studies, a schedule of three bi-weekly s.c. immunizations of 0.1 or 1 μg of the conjugate (PS content) without an adjuvant induced serum antibody levels of >10,000 units/mL measured by enzyme-linked immunosorbent assay (ELISA) as compared to ∼100 units/mL in PS control mice. The elicited antibodies were active in bactericidal assays using either baby rabbit or human complement (titers >1500 compared to ∼200 for the PS control group). The synthesis process is reproducible and scalable, and has been successfully used for manufacturing a Mn A PS–TT conjugate vaccine based on a paradigm of shared manufacturing with transfer of new technology [Jodar L, LaForce FM, Ceccarini C, Aguado T, Granoff DM. Meningococcal conjugate vaccine for Africa: a model for development of new vaccine for the poorest countries. Lancet 2003, 361:1092–4]. A phase 1 clinical trial of the manufactured Men A–TT conjugate vaccine has been successfully carried out in adults in India, and a phase 2 clinical trial in young children is currently underway in Africa.     

Cationic microparticles are a potent delivery system for a HCV DNA vaccine

We initially evaluated in mice the ability of naked DNA encoding intracellular forms of the E1E2 envelope proteins from HCV to induce antibody responses and compared the responses induced with the same plasmid adsorbed onto cationic poly (lactide co-glycolide) (PLG) microparticles. Although naked DNA was only able to induce detectable responses at the 100 microg dose level, making this approach impractical for evaluation in larger animals, PLG/DNA induced detectable responses at 10 microg. In addition, the PLG/DNA microparticles induced significantly enhanced responses to naked DNA when compared at the same dose level. Remarkably, PLG/DNA induced comparable responses to recombinant E1E2 protein adjuvanted with the emulsion MF59. Furthermore, PLG/DNA effectively primed for a booster response with protein immunization, while naked DNA did not. Therefore, PLG/DNA was selected for further evaluation in a non-human primate model. In a study in rhesus macaques, PLG/DNA induced seroconversion in 3/3 animals following three immunizations. Although the antibody responses appeared lower than those induced with recombinant protein adjuvanted with MF59, following a fourth dose, PLG/DNA and protein induced comparable responses. However, a single booster dose of recombinant protein administered to the animals previously immunized with PLG/DNA induced much higher responses. In addition, one of three animals immunized with PLG/DNA showed a cytotoxic T lymphocyte response in peripheral blood lymphocytes. In conclusion, cationic PLG microparticles with adsorbed HCV DNA generates potent immune responses.     

Parameters affecting the immunogenicity of microencapsulated tetanus toxoid.

The immunogenic potential of tetanus toxoid (TT) was compared when either adsorbed to aluminium hydroxide (TT-alum) or entrapped in microparticles consisting of poly(D,L-lactic-co-glycolic acid) (PLA:PGA, 55:45) derived polymers. Furthermore, the effect of administering the microparticles in an aqueous buffer or water-in-oil emulsion on the TT immunogenicity was also investigated. When mice were immunized with the different formulations, similar levels of anti-TT antibodies were observed during the primary IgG response. The choice of the carrier seemed to play an important role for both the level and maintenance of the secondary IgG response, attained as a consequence of a booster immunization with TT-alum. The strongest secondary antibody response was obtained by priming with TT-containing microparticles, resuspended in water-in-oil emulsions. As expected, incomplete Freund's adjuvant (IFA) proved to be a more potent adjuvant than peanut oil, whereas resuspension of the microparticles in aqueous solution induced a relatively less efficient antibody response. Overall, microencapsulated TT primed the mice more effectively, since the secondary antibody response was higher and persisted longer compared with TT-alum priming. These results indicate that in addition to TT maintaining its antigenicity after microencapsulation, the microparticles also potentiate its immunogenic properties. This approach should prove very useful for designing more effective vaccines.     

Protection against Bordetella pertussis infection following parenteral or oral immunization with antigens entrapped in biodegradable particles: effect of formulation and route of immunization on induction of Th1 and Th2 cells

The immunogenicity and protective efficacy of systemically and orally delivered pertussis antigens entrapped in either microparticle poly-lactide-co-glycolide (PLG) or nanoparticle PLG formulations were evaluated in a murine respiratory challenge model for infection with Bordetella pertussis. The results demonstrate that immunization with two parenteral doses of 1 microg or three oral doses of 100 microg of pertussis toxoid (PTd) and filamentous haemagglutinin (FHA) encapsulated in PLG conferred a high level of protection against B. pertussis challenge. Furthermore protection could be generated with a single parenteral immunization with a combined microparticle and nanoparticle formulation. However, the route of immunization and the size of the particles affected the type of T cell response induced. Parenteral immunization with PTd and FHA entrapped in PLG microparticles elicits a potent type 1 T cell response and potent antibody response when given by the intraperitoneal (i.p.) or intramuscular (i.m.) route. In contrast, nanoparticle formulations favoured the induction of Th2 cells.     

Mycobacterium tuberculosis arabinomannan-protein conjugates protect against tuberculosis

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.     

Safety of MF59 adjuvant

The need to enhance the immunogenicity of purified subunit antigens has prompted the development of new adjuvants. The adjuvant emulsion MF59 has been tested in animals in combination with different antigens and finally evaluated in humans. It was licensed after the successful outcome of preclinical and clinical testing. This paper summarizes the main characteristics of the MF59 adjuvant, including animal testing, clinical experience with various vaccines, and information from current postmarketing surveillance data. This review supports the hypothesis that MF59 is a safe adjuvant for human use.     

Recombinant hemagglutinin produced from Chinese Hamster Ovary (CHO) stable cell clones and a PELC/CpG combination adjuvant for H7N9 subunit vaccine development

The novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development.     

Immunogenicity and lower dose requirement of polymer entrapped tetanus toxoid co-administered with alum

Low adjuvanticity of microparticles based vaccine formulation necessitates the use of alum along with particles to elicit improved antibody titers from single point immunization. It was observed that antibody response from immunization with admixture of alum and polymer entrapped antigen was dependent on particle size, amount of antigen released during burst phase and dose of microencapsulated antigen. In the animals immunized with polymer entrapped tetanus toxoid (TT) very large particles (50-150 microm) did not elicited high antibody titers where as microparticles in the range 2-8 microm exhibited remarkable improvement in the antibody response. Very small size particles (<2 microm) were also not as effective as 2-8 microm size particles for generation of antibody response. Presence of alum improved the immune response by adsorbing the burst released antigen from the particle surfaces. Role of alum in potentiation of immune response from polymer entrapped TT was highly significant at lower dose regimes. Polymer entrapped TT as little as 0.1 Lf when immunized along with alum generated antibody responses superior to those elicited by 10 Lf soluble antigen. Immunization with admixture of particles and alum generated two to three times higher antibody titer than that observed from immunization with similar single doses of alum adsorbed TT. Single point immunization of admixture of particles entrapped TT and alum generated sustained long lasting antibody responses comparable to two divided doses of alum-adsorbed antigen. Superiority of single dose polymeric formulation in comparison to two divided doses of alum adsorbed TT was more evident at lower doses of TT immunization. This reflected the profound synergistic effect of both the adjuvants at lower doses. Affinity of antibodies generated from single point immunization was comparable to that achieved with two doses of alum adsorbed TT immunization. Particle alone elicited more of IgG2a type antibody where as immunization with admixture of alum and particles improved the overall antibody response and more of Th2 type response.     

Tolerability and immunogenicity of an 11-valent pneumococcal conjugate vaccine in adults

We studied the safety and immunogenicity in healthy adults of an 11-valent pneumococcal conjugate vaccine. Capsular polysaccharides (PS) of serotypes 1, 4, 5, 7F, 9V, 19F and 23F were conjugated to tetanus toxoid, and of serotypes 3, 6B, 14 and 18C to diphtheria toxoid. Ten subjects received the conjugate vaccine with and the other ten subjects without aluminium hydroxide adjuvant. The reference vaccine was a marketed 23-valent PS vaccine. Safety data were recorded over 5 days after the immunisation. IgG antibody concentrations, avidity and subclass distribution were measured by EIA. The conjugate without aluminium induced more local adverse effects than the conjugate with aluminium or PS vaccine. All vaccines evoked significant antibody increases to all vaccine specific antigens. Both conjugate vaccines induced antibodies mainly of IgG(2) subclass, and adjuvanted conjugate vaccine induced IgG antibodies with increased avidity. This first administration, to man, of a mixed protein carrier 11-valent pneumococcal conjugate vaccine demonstrated its ability to induce an immune response without significant adverse effects, enabling further study on its use in paediatric populations.     

RLJ-NE-299A: a new plant based vaccine adjuvant

Alum has been in use since long as an adjuvant for vaccines. However, its use as a vaccine adjuvant offers limitation in supporting cell mediated response. Therefore, a new plant based product RLJ-NE-299A from Picrorhiza kurroa reported for its immunostimulatory activity, has been explored for its potential as an alternative adjuvant. In order to compare the adjuvant activity with alum, antigen-specific immune responses were evaluated following immunization with a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with RLJ-NE-299A and alum in mice. The adjuvant RLJ-NE-299A up-regulated remarkably the expression of Th1 cytokines IL-2, IL-12, IFN-gamma, TNF alpha and Th2 cytokine IL-4 in lymph node cell cultures after 2 weeks of primary immunization with HBsAg. Further, the levels of both immunoglobulins IgG2a (Th1) and IgG1 (Th2) subtypes increased profoundly in blood sera of mice immunized with HBsAg/RLJ-NE-299A. The results indicated that RLJ-NE-299A has strong potential to increase both cell mediated and humoral immune responses and is capable of sustaining the total antigen-specific antibody response. Besides, the RLJ-NE-299A provides a signal to gear up both CD4 helper cells (Th1 and Th2) and CD8 cells populations, which may have important implications for vaccination against hepatitis B virus. Variable doses of RLJ-NE-299A (0.312-40 microg) containing vaccine antigen (HBsAg) were well tolerated with optimum T cell response at 2.5 microg/ml. Not only this, the adjuvant was also able to induce cellular immune responses to HBsAg as evidenced by Th1 and Th2 cytokines upregulation, which enabled mice to overcome the unresponsiveness to antigen HBsAg encountered with alum-adjuvanted vaccine in otherwise non-responding mice population. The study presents evidence that the HPLC standardized fraction RLJ-NE-299A, is an adjuvant of choice over alum in improving and maintaining the improved immune status against HBsAg, and may also prove useful adjuvant candidate with other vaccine antigens, too.     

Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes

Vaccine adjuvants such as alum and the oil-in-water emulsion MF59 are used to enhance immune responses towards pure soluble antigens, but their mechanism of action is still largely unclear. Since most adjuvanted vaccines are administered intramuscularly, we studied immune responses in the mouse muscle and found that both adjuvants were potent inducers of chemokine production and promoted rapid recruitment of CD11b⁺ cells. The earliest and most abundantly recruited cell type are neutrophils, followed by monocytes, eosinophils and later dendritic cells (DCs) and macrophages. Using fluorescent forms of MF59 and ovalbumin (OVA) antigen, we show that all recruited cell types take up both adjuvant and antigen to transport them to the draining lymph nodes (LNs). There, we found antigen-positive neutrophils and monocytes within hours of injection, later followed by B cells and DCs. Compared to alum, MF59-injection lead to a more prominent neutrophil recruitment and a more efficient antigen re-localization from the injection site to the LN. As antigen-transporting neutrophils were observed in draining LNs, we asked whether these cells play an essential role in MF59-mediated adjuvanticity. However, antibody-mediated neutrophil ablation left MF59-adjuvanticity unaltered. Further studies will reveal whether other single cell types are crucial or whether the different recruited cell populations are redundant with overlapping functions.     

Mixing is required for uniform reconstitution of filter-dried protein antigens in a single-injection vaccine formulation

Ambient temperature filter dried vaccine formulations have been proposed to simultaneously achieve thermostability and offer a ready-to-use immunisation device that combines reconstitution and injection. Vaccine concentration should be uniform at the point of injection, but the uniformity following direct reconstitution of filter-dried vaccines has not been reported. We present here a study of vaccine mixing and release following dissolution of filter-dried model protein and toxoid antigens within a single syringe, filter and needle unit. Release was better for filters made from glass than cellulose. Without additional mixing, uniformity was poor and only 41% of input protein was released from protein filter-dried onto glass fiber. In contrast, adding a simple glass bead and mixing by inversion, 100% release antigen solution was achieved, with uniform concentration at exit from the needle throughout a simulated injection. Adsorption onto alum adjuvant had no detectable effect on vaccine dissolution and mixing. The uniformity and yield of low doses of diphtheria and tetanus toxoid was also improved by mixing, albeit with a lower yield of 60–68%. We conclude that uniformity and mixing should be studied to ensure safety and efficacy of directly reconstituted filter-dried vaccine formulations.     

A β-mannan trisaccharide conjugate vaccine aids clearance of Candida albicans in immunocompromised rabbits

A β-(1→2)-linked mannose trisaccharide epitope that is the optimal inhibitor of two protective monoclonal antibodies specific for the Candida albicans cell wall phosphomannan was used to create a synthetic conjugate vaccine. Two injections of the trisaccharide-tetanus toxoid conjugate administered with alum induced a robust secondary antibody response in rabbits with trisaccharide specific IgG ELISA titers in excess of 1:100,000. Fluorescent labeling studies demonstrated these antibodies (i) recognized the cell wall β-mannan of C. albicans on hyphae and budding cells and (ii) C. albicans incubated with immune sera bound complement factor C3. The synthetic conjugate vaccine but not the carrier protein, tetanus toxoid reduced Candida load in vaccinated rabbits subsequently rendered leukocytopenic by injection of cyclophosphamide and then challenged with live C. albicans. These data support the contention that antibody mediated immunity plays a role in combating C. albicans infections and suggests that a surprisingly simple, readily accessible synthetic conjugate vaccine may have therapeutic potential.     

Comparison of carrier proteins to conjugate malaria transmission blocking vaccine antigens,Pfs25 and Pfs230

Malaria transmission blocking vaccines (TBV) target the sexual stage of the parasite and have been pursued as a stand-alone vaccine or for combination with pre-erythrocytic or blood stage vaccines. Our efforts to develop TBV focus primarily on two antigens, Pfs25 and Pfs230. Chemical conjugation of these poorly immunogenic antigens to carrier proteins enhances their immunogenicity, and conjugates of these antigens to Exoprotein A (EPA) are currently under evaluation in clinical trials. Nonetheless, more potent carriers may augment the immunogenicity of these antigens for a more efficacious vaccine; here, we evaluate a series of proteins to identify such a carrier. Pfs25 and Pfs230 were chemically conjugated to 4 different carriers [tetanus toxoid (TT), a recombinant fragment of tetanus toxin heavy chain (rTThc), recombinant CRM₁₉₇ produced in Pseudomonas fluorescens (CRM197) or in E. coli (EcoCRM®)] and compared to EPA conjugates in mouse immunogenicity studies. Conjugates of each antigen formulated in Alhydrogel® elicited similar antibody titers but showed differences in functional activity. At a 0.5 µg dose, Pfs230 conjugated to TT, CRM197 and EcoCRM® showed significantly higher functional activity compared to EPA. When formulated with the more potent adjuvant GLA-LSQ, all 4 alternate conjugates induced higher antibody titers as well as increased functional activity compared to the EPA conjugate. IgG subclass analysis of Pfs230 conjugates showed no carrier-dependent differences in the IgG profile. While Alhydrogel® formulations induced a Th2 dominant immune response, GLA-LSQ formulations induced a mixed Th1/Th2 response.     

Evaluation of immune responses elicited in mice against a recombinant malaria vaccine based on Plasmodium vivax Duffy binding protein

Plasmodium vivax Duffy binding protein (PvDBP) binds the Duffy blood group antigen as the obligate receptor for erythrocyte invasion. We have tested in mice the immunogenicity of recombinant P. vivax region II (PvRII), the receptor-binding domain of PvDBP, formulated with five adjuvants, namely, Montanide ISA720, AS02A, alum, QS21 and MF59. All the formulations elicited high titer antibodies, with Montanide ISA720 and AS02A yielding the highest titers followed by MF59, QS21 and alum. Sera raised against PvRII formulated with AS02A and Montanide ISA720 followed by alum were most effective at blocking PvRII binding to erythrocytes in a functional assay. Analysis of cellular immune responses indicated that all adjuvant groups induced significant interferon-gamma, with alum being the highest interferon-gamma inducer. These results suggest that recombinant PvRII formulated with human compatible adjuvants is immunogenic in small animal models and that Montanide ISA720, AS02A and alum perform better than MF59 and QS21 in terms of their ability to elicit high titer binding inhibitory antibodies.