The choice of adjuvants in Mycoplasma vaccines

The use of adjuvants in vaccine production is an important aspect of potent vaccines. This investigation was concerned with finding the most efficient adjuvants for use in Mycoplasma vaccines produced in Nigeria. Four different vaccines were produced from the Gladysdale strain of Mycoplasma mycoides subspecies mycoides. They differed depending on the type of adjuvants used. Each vaccine was used to vaccinate eight cattle using a dose of 1 ml. Two other groups of eight cattle were used as controls. One of the two groups received 1 ml dose of inactivated Gladysdale vaccine without adjuvant while the second group received 1 ml dose of saline. The number of cattle that had the peak complement fixing (CF) antibody titres of 1/80 in each group of cattle was four for vaccine containing aluminium hydroxide gel, eight for vaccine containing liquid paraffin, one for vaccine containing sodium alginate and one for vaccine without adjuvant. Seven cattle from the group vaccinated with vaccine containing Freund's incomplete adjuvant had peak CF antibody titres of 1/80 or higher. The two groups vaccinated with vaccine containing liquid paraffin and Freund's incomplete adjuvant survived challenge at 6 months post vaccination. Freund's incomplete adjuvant and liquid paraffin containing 10% Arlacel A are the most efficient adjuvants.     

Comparative immunogenecity of foot and mouth disease virus antigens in FMD-haemorrhagic septicaemia combined vaccine and FMD vaccine alone in buffalo calves

Humoral immune response was evaluated by monitoring the serum antibody titres and virus specific IgM titres against Foot and Mouth Disease (FMD) virus antigens in serum samples obtained from different groups of calves inoculated with combined vaccine or FMD vaccine alone, on 0, 7, 14, 21, 28, 42 and 56 days post-vaccination (DPV). The cellular immune response was monitored by MTT based lymphoproliferation in peripheral blood mononuclear cell cultures. Higher liquid phase blocking (LPB) ELISA antibody titres were observed in calves receiving combined vaccine as compared to calves immunized with FMD vaccine alone with the peak titres in both the groups obtained on 21 days post-vaccination. However, the virus specific IgM titres were significantly higher in group of calves inoculated with combined vaccine than FMD vaccine alone. The lymphoproliferative responses against FMDV types O, A22 and Asia 1 in the groups receiving combined vaccine and FMD vaccine alone started increasing gradually after day 14 and reached peak levels on 28 DPV followed by a gradual decline subsequently. The group receiving combined vaccine showed higher proliferative responses on in vitro stimulation with FMD virus type O, whereas, with FMD virus type Asia 1, the responses were significantly higher on 14 and 21 DPV as compared to the group immunized with FMD vaccine alone. However, in the group receiving combined vaccine, the responses on in vitro stimulation with FMD virus type A22 were significantly higher than FMD vaccine alone group on all DPV except on 42 DPV.     

Saponin Adjuvants. IV. Evaluation of The Adjuvant Quil a in the Vaccination of Cattle Against Foot-And-Mouth Disease

The saponin adjuvant Quil A has been investigated in the vaccination of cattle against foot-and-mouth disease. Using a Frenkel type vaccine a dose-response relationship has been established between Quil A and neutralizing antibody titres. Ten ml of vaccine was combined with 0, 50, 200, 800, and 3200 µg of Quil A. The combinations were each injected into 4 animals. The local reaction on the site of injection produced by injection of the vaccine alone and in combination with different doses of Quil A has been estimated. On this basis a therapeutical dose at 1 mg of Quil A has been estimated to combine maximum adjuvant effect with a minimum of adverse reactions. This dose has been tested in the vaccination of cattle with FMD vaccines derived from BHK suspension cell virus of type O and A respectively. The vaccines were tested in 10 ml and 5 ml doses with or without Quil A, and each in 4 animals. It is concluded that Quil A is a valuable adjuvant for use in the induction of neutralizing antibodies against foot-and-mouth disease in cattle.     

Effects of leptin Arg25Cys on peripheral mononuclear cell counts and antibody response to vaccination in beef cattle

A single nucleotide polymorphism (SNP) in the leptin gene that results in Arg25Cys has been associated with beef carcass quality and milk composition in dairy cattle. However, leptin (LEP) also plays a role in immune performance and hence it was important to determine whether selection based on this SNP would negatively affect immune cell numbers or antibody production. LEP c.73C>T was assessed for effects on immune cell counts and antibody titres in 27 beef cattle herds ( n  = 556). A commercial rabies vaccine had been administered to these animals. Prior to being vaccinated, counts of several important mononuclear cells (total and activated B lymphocytes, total and activated T helper and T cytotoxic, WC1 T lymphocytes and monocytes) as well as baseline serum antibody titres were determined for each animal. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and mononuclear cell types were again counted. Counts of six different cell types were significantly associated with the LEP genotype; however, no consistent patterns were observed between LEP genotype (TT, CT or CC) and peripheral blood mononuclear cell populations. Significant differences in the production of rabies antibodies in response to vaccination were observed relative to LEP genotype. Our results suggest that selection for either the C or T allele would not detrimentally impact on the measured indicators of immune function in beef cattle.     

Immune response in cattle vaccinated against rabies

In order to determine the best type of rabies vaccine to use as a booster, 78 serological samples from singly vaccinated cattle were analyzed by counterimmunoelectrophoresis technique. The animals were divided into several groups, received the first vaccine dose with modified live virus vaccine (ERA strain) and were revaccinated with inactivated virus or modified live virus vaccines. Boosters were given at 2, 4, 8, 12 and 16 weeks following first vaccination. Results showed high titres in the cases of booster with inactivated vaccine. In all cases, however, detectable antibody titres declined quickly.     

Development of a Foot-and-Mouth Disease Virus Serotype A Empty Capsid Subunit Vaccine Using Silkworm (Bombyx mori) Pupae

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae (Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID(50)). Furthermore, the 50% bovine protective dose (PD(50)) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD(50) per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases.     

Efficacy model for antibody-mediated pre-erythrocytic malaria vaccines

Antibodies to the pre-erythrocytic antigens, circumsporozoite protein (CSP), thrombospondin-related adhesive protein (TRAP) and liver-stage antigen 1, have been measured in field studies of semi-immune adults and shown to correlate with protection from Plasmodium falciparum infection. A mathematical model is formulated to estimate the probability of sporozoite infection as a function of antibody titres to multiple pre-erythrocytic antigens. The variation in antibody titres from field data was used to estimate the relationship between the probability of P. falciparum infection per infectious mosquito bite and antibody titre. Using this relationship, we predict the effect of vaccinations that boost baseline CSP or TRAP antibody titres. Assuming the estimated relationship applies to vaccine-induced antibody titres, then single-component CSP or TRAP antibody-mediated pre-erythrocytic vaccines are likely to provide partial protection from infection, with vaccine efficacy of approximately 50 per cent depending on the magnitude of the vaccine-induced boost to antibody titres. It is possible that the addition of a TRAP component to a CSP-based vaccine such as RTS,S would provide an increase in infection-blocking efficacy of approximately 25 per cent should the problem of immunological interference between antigens be overcome.     

Antibody Response and Protection Induced by Immunization with Smooth and Rough Strains in Experimental Salmonellosis

The antibody response of mice to a smooth strain of Salmonella typhimurium was shown previously to be extremely rapid and potent. As measured by the complement-mediated bactericidal reaction, it was also found to be highly specific as well as reproducible. Experiments which studied the effects of antigen type (live or heat-killed), antigen dose, and the route of immunization indicated that the most rapid and highest antibody response was achieved with live, smooth organisms injected by the intraperitoneal route. Living vaccines of rough strains of either S. typhimurium or S. enteritidis induced antibodies directed against the corresponding smooth organisms. The response to the rough strains was apparently due to antibody production rather than to the simple release of preformed natural antibody. The duration of protection conferred by the rough strain vaccines was closely correlated with the endotoxic content of the immunizing strain. Smooth heat-killed vaccines and a rough live vaccine protected against homologous but not heterologous challenge. In contrast, immunization with a smooth live vaccine protected mice against both homologous and heterologous challenge infections. Protection was not due to a local effect in the peritoneal cavity, since mice were also protected against subcutaneous challenge. The secondary antibody response, induced in immunized animals by the virulent challenge infection, was demonstrated to be rapid and potent, and hence a factor to be considered in protection.     

Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine

The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.     

Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3-5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.     

Characterization of Foot-And-Mouth Disease Viruses (FMDVs) from Ugandan Cattle Outbreaks during 2012-2013: Evidence for Circulation of Multiple Serotypes

To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012–2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered.     

The Expression of IL6 and 21 in Crossbred Calves Upregulated by Inactivated Trivalent FMD Vaccine

Foot and mouth disease (FMD) is an economically important disease and a whole-virus inactivated trivalent virus vaccine is the mainstay for controlling the disease in India. The protective humoral immune response to FMD vaccination is a complex, but, tightly regulated process mediated by the interplay of interleukins (IL). Based on the specific role of IL6 and 21 in adaptive immune response, we hypothesized that inactivated trivalent FMD vaccine would stimulate IL6 and 21 expression in the circulating lymphocytes. The expressions of IL6 and 21 were assayed on 0, 28, 60, 90, and 120 d post-vaccination (DPV) by quantitative PCR (qPCR) with simultaneous assessment of FMDV antibody titer by liquid phase blocking ELISA. The results revealed that the peak expression of IL6 and 21 was on DPV 28 which correlated well with the FMDV antibody titer and plummeted to the prevaccination titer level by 60 DPV. As IL21 is the final effector of antibody production as compared to IL6, we investigated the expression of IL21 in calves that had protective titer (>1.8) with the unprotected group (<1.8). Expression of IL21 on 28 DPV was numerically higher in the protected than that of the unprotected group of calves.     

CpG-enriched plasmid enhances the efficacy of the traditional foot-and-mouth disease killed vaccine

A CpG-enriched recombinant plasmid (pUC18-CpG) as an adjuvant of FMD killed vaccine was tested for immunization and vaccination challenge in a porcine model. Our preliminary results had indicated that the recombinant plasmid could enhance the humoral immune response triggered by the traditional oil-adjuvant vaccine after the initial inoculation. A subsequent vaccination-challenge test showed an increased PD(50) value. Thus, coadministration of the recombinant plasmid with the oil-adjuvant vaccine helped illicit an immune response earlier than that elicited by giving the vaccine alone. Our results showed that pUC18-CpG can be a potent immunoadjuvant for the traditional FMD killed vaccine and can greatly enhance the traditional vaccine's efficacy when given in combination with it.     

Molecular vaccine prepared by fusion of XCL1 to the multi-epitope protein of foot-and-mouth disease virus enhances the specific humoural immune response in cattle

Targeting antigen to dendritic cells (DCs) is a promising way to manipulate the immune response and to design prophylactic molecular vaccines. In this study, the cattle XCL1, ligand of XCR1, was fused to the type O foot-and-mouth disease virus (FMDV) multi-epitope protein (XCL-OB7) to create a molecular vaccine antigen, and an ^△XCL-OB7 protein with a mutation in XCL1 was used as the control. XCL-OB7 protein specifically bound to the XCR1 receptor, as detected by flow cytometry. Cattle vaccinated with XCL-OB7 showed a significantly higher antibody response than that to the ^△XCL-OB7 control (P < 0.05). In contrast, when XCL-OB7 was incorporated with poly (I:C) to prepare the vaccine, the antibody response of the immunized cattle was significantly decreased in this group and was lower than that in the ^△XCL-OB7 plus poly (I:C) group. The FMDV challenge indicated that cattle immunized with the XCL-OB7 alone or the ^△XCL-OB7 plus poly (I:C) obtained an 80% (4/5) clinical protective rate. However, cattle vaccinated with ^△XCL-OB7 plus poly (I:C) showed more effective inhibition of virus replication than that in the XCL-OB7 group after viral challenge, according to the presence of antibodies against FMDV non-structural protein 3B. This is the first test of DC-targeted vaccines in veterinary medicine to use XCL1 fused to FMDV antigens. This primary result showed that an XCL1-based molecular vaccine enhanced the antibody response in cattle. This knowledge should be valuable for the development of antibody-dependent vaccines for some infectious diseases in cattle.

     

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.     

Protective immune response against foot-and-mouth disease.

The causative agents of foot-and-mouth disease (FMD) are small icosahedral viruses of the Aphthovirus group within the Picornaviridae family. There is no evidence that these viruses infect cells of the immune system or otherwise interfere detrimentally with their function; additionally, it has not been possible to relate cytotoxicity reactions against virus-infected cells to the efficacy of the immune response against FMD virus infection. In contrast, there is a close association between FMD virus antibody and the protective immune response (10, 14, 15, 20, 24, 25, 29-32). Induction of this antibody is dependent on the structure of the viral antigenic sites (7-9, 11, 18) and on the concomitant presence of Th-lymphocyte epitopes (4, 5, 7, 8), although a Th-lymphocyte-independent response has been reported (2). Recent work by Piatti et al. (26) showed that the immune response induced by FMD virus was only Th-lymphocyte dependent when low doses of antigen were used. This latter work was performed in mice, and it is not certain that a similar situation would be found in cattle. As for the major effector immune defense, this relies on the interaction between antibody-virus complexes and the phagocytic cells of the reticuloendothelial system (17, 19).     

Increased Humoral Immune Responses of Pigs to Foot‐and‐Mouth Disease Vaccine Supplemented with Ginseng Stem and Leaf Saponins

Vaccination is a conventional approach against foot‐and‐mouth disease (FMD) in pigs. However, failure to elicit an immune response to vaccine has been reported. Our previous investigation showed that ginseng stem and leaf saponins (GSLS) and mineral oil acted synergistically to promote Th1/Th2 immune responses to FMD vaccine in mice. This study was designed to i) find the optimal doses of GSLS in oil‐emulsified FMD vaccines to induce immune responses in mice and pigs and ii) to evaluate the effect of oil‐emulsified FMD vaccine supplemented with GSLS on the immune responses in pigs, by measuring the serum indirect hemagglutination (IHA) titer and IgG and IgG subclass levels. The GSLS‐enhanced immune response to FMD oil‐emulsion vaccine depended on the dose of GSLS added to the vaccine. Addition of GSLS at a dose of 40 μg to 2 ml of FMD oil‐emulsified vaccine significantly enhanced the humoral immune responses in pigs, when compared to the vaccine without GSLS (P<0.05). The increased antibodies included IgG1 and IgG2. Hence, GSLS and oil adjuvant synergistically promoted the immune responses to vaccination against FMD in pigs, and GSLS could be a promising vaccine additive to improve oil‐emulsified veterinary vaccines.     

Skin vaccination against cervical cancer associated human papillomavirus with a novel micro-projection array in a mouse model

Background

Better delivery systems are needed for routinely used vaccines, to improve vaccine uptake. Many vaccines contain alum or alum based adjuvants. Here we investigate a novel dry-coated densely-packed micro-projection array skin patch (Nanopatch™) as an alternate delivery system to intramuscular injection for delivering an alum adjuvanted human papillomavirus (HPV) vaccine (Gardasil®) commonly used as a prophylactic vaccine against cervical cancer.

Methodology/Principal Findings

Micro-projection arrays dry-coated with vaccine material (Gardasil®) delivered to C57BL/6 mouse ear skin released vaccine within 5 minutes. To assess vaccine immunogenicity, doses of corresponding to HPV-16 component of the vaccine between 0.43±0.084 ng and 300±120 ng (mean ± SD) were administered to mice at day 0 and day 14. A dose of 55±6.0 ng delivered intracutaneously by micro-projection array was sufficient to produce a maximal virus neutralizing serum antibody response at day 28 post vaccination. Neutralizing antibody titres were sustained out to 16 weeks post vaccination, and, for comparable doses of vaccine, somewhat higher titres were observed with intracutaneous patch delivery than with intramuscular delivery with the needle and syringe at this time point.

Conclusions/Significance

Use of dry micro-projection arrays (Nanopatch™) has the potential to overcome the need for a vaccine cold chain for common vaccines currently delivered by needle and syringe, and to reduce risk of needle-stick injury and vaccine avoidance due to the fear of the needle especially among children.     

Comparison of hemagglutination inhibition and hemagglutinin pseudovirus neutralization titres in relation to protection against influenza in a mouse model

The hemagglutination inhibition (HI) test has long been used as a standard measure of antibody response for inactivated influenza vaccines. However, the HI test has limitations, such as insensitivity when using some H3N2 virus strains and failure to detect neutralizing antibodies that target regions distant from the receptor binding site. We therefore examined a hemagglutinin pseudovirus neutralization (PVN) test as a possible supplement or alternative to the HI test. We evaluated the association of HI or PVN titres with protection against influenza infection in mice based on morbidity (where the illness was defined as 25% body weight loss). We assessed this relationship using dose–response models incorporating HI or PVN titres as a variable. The morbidity was correlated with the pre-exposure titres, and such a correlation was well described by a modified dose–response model. The mathematical modelling suggests that PVN titres consistently show a stronger association with in vivo protection as compared to HI titres in mice. Given our findings, the PVN test warrants further investigation as a tool for evaluating antibody responses to influenza vaccines containing hemagglutinin. The resulting models may also be useful for analyzing human clinical data to identify potentially protective antibody titres against influenza illness.     

Generation of humoral immune responses to multi-allele PfAMA1 vaccines; effect of adjuvant and number of component alleles on the breadth of response

There is increasing interest in multi-allele vaccines to overcome strain-specificity against polymorphic vaccine targets such as Apical Membrane Antigen 1 (AMA1). These have been shown to induce broad inhibitory antibodies in vitro and formed the basis for the design of three Diversity-Covering (DiCo) proteins with similar immunological effects. The antibodies produced are to epitopes that are shared between vaccine alleles and theoretically, increasing the number of component AMA1 alleles is expected to broaden the antibody response. A plateau effect could however impose a limit on the number of alleles needed to achieve the broadest specificity. Moreover, production cost and the vaccine formulation process would limit the number of component alleles. In this paper, we compare rabbit antibody responses elicited with multi-allele vaccines incorporating seven (three DiCos and four natural AMA1 alleles) and three (DiCo mix) antigens for gains in broadened specificity. We also investigate the effect of three adjuvant platforms on antigen specificity and antibody functionality. Our data confirms a broadened response after immunisation with DiCo mix in all three adjuvants. Higher antibody titres were elicited with either CoVaccine HT™ or Montanide ISA 51, resulting in similar in vitro inhibition (65–82%) of five out of six culture-adapted P. falciparum strains. The antigen binding specificities of elicited antibodies were also similar and independent of the adjuvant used or the number of vaccine component alleles. Thus neither the four extra antigens nor adjuvant had any observable benefits with respect to specificity broadening, although adjuvant choice influenced the absolute antibody levels and thus the extent of parasite inhibition. Our data confirms the feasibility and potential of multi-allele PfAMA1 formulations, and highlights the need for adjuvants with improved antibody potentiation properties for AMA1-based vaccines.