Characterization of Antibody Responses to a Plasmodium falciparum Blood-Stage Antigen Induced by a DNA Prime/Protein Boost Immunization Protocol

The humoral immune responses elicited by priming with a DNA plasmid and boosting with either the plasmid or the corresponding recombinant protein in alum adjuvant were compared. The plasmid DNA encoded a sequence (M3) derived from the Plasmodium falciparum antigen Pf155/RESA, and the recombinant protein consisted of the identical malarial sequence fused to an albumin-binding region (BB) of streptococcal protein G. Mice of different genetic backgrounds (CBA, Balb/c and C57Bl/6) were primed with plasmid DNA and boosted with either plasmid or recombinant protein. In all strains of mice, boosting with protein elicited higher anti-M3 antibody levels than obtained by boosting with plasmid, yet the kinetics and longevity of the secondary responses were comparable. Antiserum obtained after protein boosting displayed an immunoglobulin (Ig)G subclass profile skewed to the IgG1 isotype, regardless of the mouse strain. In contrast, mice receiving a second injection with plasmid responded with a more mixed IgG subclass profile. Inclusion of a P. falciparum circumsporozoite protein-derived T-helper epitope (CS.T3) in the immunization plasmid as well as in the fusion protein, did not significantly change the humoral responses to M3. The results show the potential of DNA vaccination for the purpose of priming an antibody response against the malarial blood-stage antigen Pf155/RESA. When combined with a protein boost, this DNA priming results in high-titred and long-lasting anamnestic responses.     

New Candidate Vaccines against Blood-Stage Plasmodium falciparum Malaria: Prime-Boost Immunization Regimens Incorporating Human and Simian Adenoviral Vectors and Poxviral Vectors Expressing an Optimized Antigen Based on Merozoite Surface Protein 1

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.Attempts to generate protective blood-stage immunity to Plasmodium falciparum by vaccination in humans have met with limited success to date (18). The focus for most vaccine candidates has been on the induction of antibodies against merozoite antigens and merozoite surface protein 1 (MSP-1) in particular (24). Antibodies against the blood stage of P. falciparum are known to contribute to protective immunity in humans (40). However, the induction of antibodies to the 42-kDa portion of MSP-1 (MSP-1₄₂) appeared to be insufficient to provide protective immunity in humans in one study (39). Evidence from both animal models and humans (detailed below) suggests that cell-mediated immune responses to MSP-1 could be additionally required to induce protective immune responses.During the process of merozoite invasion into erythrocytes, MSP-1 undergoes two proteolytic processing steps; following the first step, only MSP-1₄₂ remains membrane bound, and a second cleavage of MSP-1₄₂ into 33-kDa (MSP-1₃₃) and 19-kDa (MSP-1₁₉) portions is then required for erythrocyte invasion (4). MSP-1₁₉ is a major target of protective antibodies, and MSP-1₃₃ is a target of both CD8⁺ T cells and CD4⁺ helper T cells (11, 21, 25). Antibodies to MSP-1₁₉ are thought to act though the direct inhibition of merozoite invasion into the red blood cell and via cytophilic antibody-mediated antibody-dependent cellular inhibition (24, 33). CD4⁺ T cells specific to MSP-1₃₃ are able to partially protect nude mice from lethal Plasmodium chabaudi and Plasmodium yoelii infections (53, 57), while transferred antibodies to MSP-1₁₉ alone are unable to protect nude mice against P. yoelii (22). CD4⁺ T cells against MSP-1₃₃ play an important role in providing help for priming MSP-1₁₉-specific B cells in vaccine-induced protection against murine malaria (11), and depletion of CD4⁺ T cells has been shown to reduce protection against P. yoelii (23).Following the discovery that MSP-1 is also expressed late in the liver stage (49), CD8⁺ T cells directed against MSP-1₃₃ have been shown to protect against P. yoelii in the preerythrocytic stage (11, 27). In addition, immune responses induced by immunization with nonlethal blood-stage parasites of P. yoelii have been shown to protect against sporozoite challenge, through CD4⁺ and CD8⁺ T cell mechanisms and at least partly through release of gamma interferon (IFN-γ) (2). This discovery that CD8⁺ T cells mediate significant antiparasitic activity against the liver stage of P. yoelii provides an argument that similar mechanisms may occur in human P. falciparum malaria. Further suggestion of the role of cellular immunity in protection against P. falciparum comes from those studies in humans in which protective immunity has been associated with significant cellular immune responses to blood-stage parasites, in the absence of strong blood-stage antibody responses (42, 47). In the first study, the secretion of IFN-γ appeared to be associated with protection against blood-stage P. falciparum malaria (42), and in the second, the presence of polyfunctional T cells, secreting tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) in combination with IFN-γ when stimulated by blood-stage parasites, was shown to be associated with protection against P. falciparum (47). We therefore sought to develop a vaccine targeting MSP-1, which would induce strong cellular immune responses in conjunction with high antibody titers.While inhibitory antibodies prevent MSP-1₁₉ processing and erythrocyte invasion and appear to be beneficial to the human host, blocking antibodies act to inhibit the action of these beneficial antibodies (19). Enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays (IFAs) are routinely used to quantify the responses to vaccination but give no functional information as to levels of invasion-inhibitory antibodies. Growth-inhibitory activity (GIA) assays measure the growth of P. falciparum in the presence of immune sera in vitro, and such assays may be of greater clinical relevance when assessing vaccines targeting regions of MSP-1. The definition of epitopes for inhibitory and blocking monoclonal antibodies has enabled the design of vaccines that aim to induce inhibitory, in preference to blocking, antibodies against MSP-1₁₉ (13, 16). We investigated the inclusion of this approach in the design of MSP-1 antigens in this study.Regions of MSP-1, such as MSP-1₃₃, show extensive polymorphism, and this divides those sequences into two sets of allelic families that demonstrate extensive diversification (55). Allelic variation has been shown to reduce antibody production by single-allele vaccine constructs to heterologous strains in humans (37) and to reduce protection against heterologous strain challenge in nonhuman primates (31). Moreover human T cell responses against some peptides representing these two major allelic types of MSP-1 have been found to be mutually inhibitory in in vitro assays, a phenomenon termed altered peptide ligand antagonism (28). Within the ∼190-kDa protein sequence of MSP-1, blocks have been defined on the basis of their extensive, more limited, and minimal genetic diversity (55). For example in block 8 there is only 10% homology at the amino acid level, while in block 17, encoding MSP-1₁₉, there is 90% homology (55). Blocks 16 (MSP-1₃₃) and 17 (MSP-1₁₉) together encode MSP-1₄₂.Virus-vectored vaccines are becoming established as relatively inexpensive, effective, and safe alternative vaccine platforms to conjugate protein-in-adjuvant vaccines (12, 20). We have previously demonstrated the induction of high-titer protective antibodies against P. yoelii rodent malaria by using a recombinant replication-incompetent form of human adenovirus serotype 5 (AdHu5) and modified vaccinia virus Ankara (MVA) as vaccine vectors in a heterologous prime-boost regimen (13). A heterologous human adenoviral regimen has recently been shown to induce a strong, polyfunctional, and protective T cell response against simian immunodeficiency virus (SIV) in rhesus macaques (30), and a homologous AdHu5 regimen expressing MSP-1₄₂ has been shown to induce GIA in rabbits (5). Experience in the HIV vaccine field has emphasized the importance of avoiding preexisting antivector immunity when developing vectored vaccines (6). The immunogenicity of virus-vectored vaccines is reduced in the presence of preexisting vector-neutralizing antibodies against AdHu5 (6-8), but such antibodies do not reduce the immunogenicity of simian adenoviral vectors in humans (K. Ewer et al. unpublished data). We hypothesized that a heterologous chimpanzee adenoviral vector regimen may induce strong immune responses and be more suitable for clinical use. The simian adenoviruses C6, C7, and C9 have structural similarity and sequences close to those of human adenovirus 4 in subgroup E. These vaccine vectors have shown promise in preclinical vaccines against infections such as rabies and HIV (45, 59). An alternative simian adenovirus, AdCh63, which is closely related to C6, C7, and C9, has recently been found to be safe, immunogenic, and efficacious for human use when used to express the well-studied preerythrocytic vaccine candidate antigen ME-TRAP (Ewer et al., submitted for publication). We therefore assessed our MSP-1 vaccine constructs in simian adenovirus-vectored vaccines.To develop an MSP-1 vaccine for clinical trials, we investigated the possibility of including in vectors (i) the conserved blocks of MSP-1, (ii) both allelic forms of MSP-1₄₂, and (iii) a modified MSP-1 sequence to improve antibody fine specificity and of using a variety of DNA-based virus-vectored vaccines, including several simian adenoviral serotypes. We show in animal models that both cellular and humoral immune responses can be generated by this approach. We propose that such responses may help to overcome some of the limitations of previous generations of vaccines against MSP-1.     

恶性疟原虫抗原—痘苗病毒重组活疫苗株的实验室评价

以恶性疟原虫保护性抗原复合基因—痘苗病毒重组活疫苗候选株为研究对象 ,探索其免疫血清及IgG的体外抗疟原虫能力、保护性细胞免疫反应及换人血猕猴模型动物抗攻击能力试验。结果表明 ,受检免疫血清及IgG具有一定的体外抑制恶性疟原虫增殖作用。家兔及大白鼠免疫后 4～ 6周血清中可产生明显的IL 2活性 ,免疫后 6周家兔、大白鼠及小白鼠血清IFN的活性水平比免疫前明显升高。免疫后 1个月攻虫 ,免疫猴从第 3天一直到第 12天血检中均未发现疟原虫 ;而非重组痘苗病毒免疫猴第 3天后原虫感染率上升 ,第 6天原虫感染率最高达到 6 0 % ,而后原虫感染率逐渐下降 ,持续 13d ;空白对照猴第 3天后原虫感染率也上升 ,第 8天原虫感染率最高达到 2 5 % ,而后原虫感染率逐渐下降 ,持续 12d。结果初步表明该候选疫苗株具有一定的诱发体液免疫、细胞免疫反应及抗虫体攻击能力 ,值得做进一步的研究。     

The Transferrin Binding Protein B of Moraxella catarrhalis Elicits Bactericidal Antibodies and Is a Potential Vaccine Antigen

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.     

Opsonization and Phagocytosis of Plasmodium falciparum Merozoites Measured by Flow Cytometry

A flow cytometric phagocytosis assay was established to investigate the role of anti-merozoite antibody, complement, and cytokines on the phagocytosis of Plasmodium falciparum merozoites by human neutrophils. This assay involved allowing fluorescein isothiocyanate-labeled merozoites to interact with phagocytes and analysis of the cells on a FACScan with Lysis II software. To differentiate the proportion of neutrophil surface-bound merozoites from the merozoites ingested by neutrophils, the fluorescence of bound merozoites was quenched by adding trypan blue. The data showed that sera from malaria-immune individuals in the Solomon Islands and Papua New Guinea promoted merozoite engulfment by neutrophils. The cytokines tumor necrosis factor alpha, gamma interferon, granulocyte-macrophage colony-stimulating factor, and interleukin-1β significantly increased the amount and the rate of merozoite phagocytosis by neutrophils. Optimum merozoite phagocytosis occurred when both cytokines and anti-malarial antibody were present.     

The Fimbrial Protein FlfA from Gallibacterium anatis Is a Virulence Factor and Vaccine Candidate

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production worldwide. Widespread multidrug resistance largely prevents treatment of this organism using traditional antimicrobial agents, while antigenic diversity hampers disease prevention by classical vaccines. Thus, insight into its pathogenesis and knowledge about important virulence factors is urgently required. A key event during the colonization and invasion of mucosal surfaces is adherence, and recently, at least three F17-like fimbrial gene clusters were identified in the genomes of several G. anatis strains. The objective of this study was to characterize the putative F17-like fimbrial subunit protein FlfA from G. anatis 12656-12 and determine its importance for virulence. In vitro expression and surface exposure of FlfA was demonstrated by flow cytometry and immunofluorescence microscopy. The predicted function of FlfA as a fimbrial subunit protein was confirmed by immunogold electron microscopy. An flfA deletion mutant (ΔflfA) was generated in G. anatis 12656-12, and importantly, this mutant was significantly attenuated in the natural chicken host. Furthermore, protection against G. anatis 12656-12 could be induced by immunizing chickens with recombinant FlfA. Finally, in vitro expression of FlfA homologs was observed in a genetically diverse set of G. anatis strains, suggesting the potential of FlfA as a serotype-independent vaccine candidate This is the first study describing a fimbrial subunit protein of G. anatis with a clear potential as a vaccine antigen.     

Antibodies Reactive with the N-Terminal Domain of Plasmodium falciparum Serine Repeat Antigen Inhibit Cell Proliferation by Agglutinating Merozoites and Schizonts

The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum. Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.     

Protective Efficacy of a Toxoplasma gondii Rhoptry Protein 13 Plasmid DNA Vaccine in Mice

Toxoplasma gondii is an obligate intracellular parasite infecting humans and other warm-blooded animals, resulting in serious public health problems and economic losses worldwide. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In the present study, a DNA vaccine expressing rhoptry protein 13 (ROP13) of T. gondii inserted into eukaryotic expression vector pVAX I was constructed, and the immune protection it induced in Kunming mice was evaluated. Kunming mice were immunized intramuscularly with pVAX-ROP13 and/or with interleukin-18 (IL-18). Then, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged with the virulent T. gondii RH strain (type I) and the cyst-forming PRU strain (type II). The results showed that pVAX-ROP13 alone or with pVAX/IL-18 induced a high level of specific anti-T. gondii antibodies and specific lymphocyte proliferative responses. Coinjection of pVAX/IL-18 significantly increased the production of gamma interferon (IFN-γ), IL-2, IL-4, and IL-10. Further, challenge experiments showed that coimmunization of pVAX-ROP13 with pVAX/IL-18 significantly (P < 0.05) increased survival time (32.3 ± 2.7 days) compared with pVAX-ROP13 alone (24.9 ± 2.3 days). Immunized mice challenged with T. gondii cysts (strain PRU) had a significant reduction in the number of brain cysts, suggesting that ROP13 could trigger a strong humoral and cellular response against T. gondii cyst infection and that it is a potential vaccine candidate against toxoplasmosis, which provided the foundation for further development of effective vaccines against T. gondii.     

Inhibitory Humoral Responses to the Plasmodium falciparum Vaccine Candidate EBA-175 Are Independent of the Erythrocyte Invasion Pathway

Plasmodium falciparum utilizes multiple ligand-receptor interactions for invasion. The invasion ligand EBA-175 is being developed as a major blood-stage vaccine candidate. EBA-175 mediates parasite invasion of host erythrocytes in a sialic acid-dependent manner through its binding to the erythrocyte receptor glycophorin A. In this study, we addressed the ability of naturally acquired human antibodies against the EBA-175 RII erythrocyte-binding domain to inhibit parasite invasion of ex vivo isolates, in relationship to the sialic acid dependence of these parasites. We have determined the presence of antibodies to the EBA-175 RII domain by enzyme-linked immunosorbent assay (ELISA) in individuals from areas of Senegal where malaria is endemic with high and low transmission. Using affinity-purified human antibodies to the EBA-175 RII domain from pooled patient plasma, we have measured the invasion pathway as well as the invasion inhibition of clinical isolates from Senegalese patients in ex vivo assays. Our results suggest that naturally acquired anti-EBA-175 RII antibodies significantly inhibit invasion of Senegalese parasites and that these responses can be significantly enhanced through limiting other ligand-receptor interactions. However, the extent of this functional inhibition by EBA-175 antibodies is not associated with the sialic acid dependence of the parasite strain, suggesting that erythrocyte invasion pathway usage by parasite strains is not driven by antibodies targeting the EBA-175/glycophorin A interaction. This work has implications for vaccine design based on the RII domain of EBA-175 in the context of alternative invasion pathways.     

Cloning and Expression of a 48-Kilodalton Babesia caballi Merozoite Rhoptry Protein and Potential Use of the Recombinant Antigen in an Enzyme-Linked Immunosorbent Assay

A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.     

Adhesion Protein ApfA of Actinobacillus pleuropneumoniae Is Required for Pathogenesis and Is a Potential Target for Vaccine Development

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia, which causes serious economic losses in the pig farming industry worldwide. Due to a lack of knowledge of its virulence factors and a lack of effective vaccines able to confer cross-serotype protection, it is difficult to place this disease under control. By analyzing its genome sequences, we found that type IV fimbrial subunit protein ApfA is highly conserved among different serotypes of A. pleuropneumoniae. Our study shows that ApfA is an adhesin since its expression was greatly upregulated (135-fold) upon contact with host cells, while its deletion mutant attenuated its capability of adhesion. The inactivation of apfA dramatically reduced the ability of A. pleuropneumoniae to colonize mouse lung, suggesting that apfA is a virulence factor. Purified recombinant ApfA elicited an elevated humoral immune response and conferred robust protection against challenges with A. pleuropneumoniae serovar 1 strain 4074 and serovar 7 strain WF83 in mice. Importantly, the anti-ApfA serum conferred significant protection against both serovar 1 and serovar 7 in mice. These studies indicate that ApfA promotes virulence through attachment to host cells, and its immunogenicity renders it a promising novel subunit vaccine candidate against infection with A. pleuropneumoniae.     

Evaluation of RevA,a Fibronectin-Binding Protein of Borrelia burgdorferi,as a Potential Vaccine Candidate for Lyme Disease

Previous studies indicated that the Lyme disease spirochete Borrelia burgdorferi expresses the RevA outer surface protein during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA appears to be a good target for preventive therapies. RevA proteins are highly conserved across all Lyme borreliae, and antibodies against RevA protein are cross-reactive among RevA proteins from diverse strains. Mice infected with B. burgdorferi mounted a rapid IgM response to RevA, followed by a strong IgG response that generally remained elevated for more than 12 months, suggesting continued exposure of RevA protein to the immune system. RevA antibodies were bactericidal in vitro. To evaluate the RevA antigen as a potential vaccine, mice were vaccinated with recombinant RevA and challenged with B. burgdorferi by inoculation with a needle or by a tick bite. Cultured tissues from all treatment groups were positive for B. burgdorferi. Vaccinated animals also appeared to have similar levels of B. burgdorferi DNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect against Borrelia burgdorferi infection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible utility of RevA-based immunotherapeutics or vaccine.     

ZnuD,a Potential Candidate for a Simple and Universal Neisseria meningitidis Vaccine

Neisseria meningitidis serogroup B (MenB) is a major cause of bacterial sepsis and meningitis, with the highest disease burden in young children. Available vaccines are based on outer membrane vesicles (OMVs) obtained from wild-type strains. However, particularly in toddlers and infants, they confer protection mostly against strains expressing the homologous protein PorA, a major and variable outer membrane protein. In the quest for alternative vaccine antigens able to provide broad MenB strain coverage in younger populations, but potentially also across all age groups, ZnuD, a protein expressed under zinc-limiting conditions, may be considered a promising candidate. Here, we have investigated the potential value of ZnuD and show that it is a conserved antigen expressed by all MenB strains tested except for some strains of clonal complex ST-8. In mice and guinea pigs immunized with ZnuD-expressing OMVs, antibodies were elicited that were able to trigger complement-mediated killing of all the MenB strains and serogroup A, C, and Y strains tested when grown under conditions of zinc limitation. ZnuD is also expressed during infection, since anti-ZnuD antibodies were detected in sera from patients. In conclusion, we confirm the potential of ZnuD-bearing OMVs as a component of an effective MenB vaccine.     

A Chimeric Plasmodium falciparum Merozoite Surface Protein Vaccine Induces High Titers of Parasite Growth Inhibitory Antibodies

The C-terminal 19-kDa domain of Plasmodium falciparum merozoite surface protein 1 (PfMSP1₁₉) is an established target of protective antibodies. However, clinical trials of PfMSP1₄₂, a leading blood-stage vaccine candidate which contains the protective epitopes of PfMSP1₁₉, revealed suboptimal immunogenicity and efficacy. Based on proof-of-concept studies in the Plasmodium yoelii murine model, we produced a chimeric vaccine antigen containing recombinant PfMSP1₁₉ (rPfMSP1₁₉) fused to the N terminus of P. falciparum merozoite surface protein 8 that lacked its low-complexity Asn/Asp-rich domain, rPfMSP8 (ΔAsn/Asp). Immunization of mice with the chimeric rPfMSP1/8 vaccine elicited strong T cell responses to conserved epitopes associated with the rPfMSP8 (ΔAsn/Asp) fusion partner. While specific for PfMSP8, this T cell response was adequate to provide help for the production of high titers of antibodies to both PfMSP1₁₉ and rPfMSP8 (ΔAsn/Asp) components. This occurred with formulations adjuvanted with either Quil A or with Montanide ISA 720 plus CpG oligodeoxynucleotide (ODN) and was observed in both inbred and outbred strains of mice. PfMSP1/8-induced antibodies were highly reactive with two major alleles of PfMSP1₁₉ (FVO and 3D7). Of particular interest, immunization with PfMSP1/8 elicited higher titers of PfMSP1₁₉-specific antibodies than a combined formulation of rPfMSP1₄₂ and rPfMSP8 (ΔAsn/Asp). As a measure of functionality, PfMSP1/8-specific rabbit IgG was shown to potently inhibit the in vitro growth of blood-stage parasites of the FVO and 3D7 strains of P. falciparum. These data support the further testing and evaluation of this chimeric PfMSP1/8 antigen as a component of a multivalent vaccine for P. falciparum malaria.     

Use of Immunodampening To Overcome Diversity in the Malarial Vaccine Candidate Apical Membrane Antigen 1

Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli, refolded in vitro, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.     

Evaluation of the Proline-Rich Antigen of Coccidioides immitis as a Vaccine Candidate in Mice

We have expressed the proline-rich antigen (PRA) from Coccidioides immitis in Escherichia coli and evaluated its potential as a vaccine candidate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant protein (rPRA) revealed two bands, which exhibited virtually identical primary amino acid sequences. T cells from rPRA-immunized BALB/c mice showed a significant in vitro proliferative response to rPRA. A small but statistically significant proliferative response was also induced by rPRA in T cells from mice immunized with whole-cell coccidioidal vaccines. BALB/c mice immunized with rPRA and challenged intraperitoneally with virulent C. immitis had a greatly reduced fungal burden in their lungs and spleens compared to unvaccinated mice. The number of organisms in the lungs was reduced 500-fold, and similar reductions were observed in the spleens of immunized mice. These studies support the continued development of rPRA as a candidate vaccine for prevention of coccidioidomycosis.     

The Th1 Immune Response to Plasmodium falciparum Circumsporozoite Protein Is Boosted by Adenovirus Vectors 35 and 26 with a Homologous Insert

The most advanced malaria vaccine, RTS,S, is comprised of an adjuvant portion of the Plasmodium falciparum circumsporozoite (CS) protein fused to and admixed with the hepatitis B virus surface antigen. This vaccine confers short-term protection against malaria infection, with an efficacy of about 50%, and induces particularly B-cell and CD4⁺ T-cell responses. In the present study, we tested by the hypothesis that the Th1 immune response to CS protein, in particular the CD8⁺ T-cell response, which is needed for strong and lasting malaria immunity, is boosted to sustainable levels vectors adenovirus and 26 with an homologous insert 35 (Ad35.CS/Ad26.CS). In this study, we evaluated immune responses induced with vaccination regimens based on an adjuvant-containing, yeast-produced complete CS protein followed by two recombinant low-seroprevalence adenoviruses expressing P. falciparum CS antigen, Ad35.CS (subgroup B) and Ad26.CS (subgroup D). Our results show that (i) the yeast (Hansenula polymorpha)produced, adjuvanted full-length CS protein is highly potent in inducing high CS-specific humoral responses in mice but produces poor T-cell responses, (ii) the Ad35.CS vector boosts the gamma interferon-positive (IFN-γ⁺) CD8⁺ T-cell response induced by the CS protein immunization and shifts the immune response toward the Th1 type, and (iii) a three-component heterologous vaccination comprised of a CS protein prime followed by boosts with Ad35.CS and Ad26.CS elicits an even more robust and sustainable IFN-γ⁺ CD8⁺ T-cell response than one- or two-component regimens. The Ad35.CS/Ad26.CS combination boosted particularly the IFN-γ⁺ and tumor necrosis factor alpha-positive (TNF-α⁺) T cells, confirming the shift of the immune response from the Th2 type to the Th1 type. These results support the notion of first immunizations of infants with an adjuvanted CS protein vaccine, followed by a booster Ad35.CS/Ad26.CS vaccine at a later age, to induce lasting protection against malaria for which the Th1 response and immune memory is required.Almost 40 years after the feasibility of vaccination against malaria was first demonstrated by means of irradiated sporozoites (9), a vaccine modality that efficiently induces long-lived protective immunity remains elusive. The most advanced circumsporozoite (CS)-based malaria vaccine candidate to date is RTS,S, a vaccine based on a fragment of Plasmodium falciparum circumsporozoite (CS) protein fused to and admixed with hepatitis B virus surface protein. In adults, RTS,S with the adjuvant AS02 has consistently conferred 40% protection against malaria infection upon sporozoite challenge (54). Even though RTS,S/AS02 induces high-level CS-specific antibody responses, the induced T-cell responses are weak (21). As the Th1 response, particularly gamma interferon (IFN-γ) and CD8⁺ T cells, is associated with protection, novel adjuvant systems were developed with the aim of improving the induced T-cell response while maintaining potent levels of CS-specific antibody responses. One of these novel adjuvant systems, AS01, demonstrated its suitability in mice, as it improved CS-specific CD4⁺ T-cell responses and led to induction of CD8⁺ T cells (32). Nonhuman primate studies also demonstrated that RTS,S with AS01 adjuvant induces strong CS-specific antibody responses as well as mean higher frequencies of IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing CD4⁺ T cells than those generated by RTS,S with AS02 adjuvant. However, the induction of CD8⁺ T cells was not confirmed in this nonhuman primate study (32). In humans, RTS,S/AS01 has been shown to induce high titers of CS-specific antibodies and higher numbers of Th1 CD4⁺ T cells than those generated by RTS,S/AS02 but no CS-specific CD8⁺ T cells (22). However, RTS,S/AS01 was able to afford 50% protection against malaria infection in adults upon sporozoite challenge (22) and 53% efficacy against disease in children between the ages of 5 and 17 months (5). These results, albeit far from being optimal, supported the progress of RTS,S/AS01 to phase III clinical trial testing in early 2009, and these trials enrolled children at the age of 6 weeks to 17 months at multiple sites in sub-Saharan Africa. It is anticipated that RTS,S/AS01 will be the first licensed malaria vaccine, provided its efficacy is confirmed in the phase III trial.Although our understanding about the correlate(s) of protection for malaria is limited, there is ample evidence that CS protein-specific antibodies, CD8⁺ T cells, and Th1 cytokines, particularly IFN-γ, play a central role in controlling the preerythrocytic and early liver stages of malaria (19, 20, 35, 47, 57). Adenovirus (Ad) vectors are particularly suited for induction of IFN-γ-producing CD8⁺ T cells required to combat malaria infection (33, 43), due to intracellular expression of a transgene inserted in the vector genome and efficient routing of expressed protein toward the class I presentation pathway. Recently, we demonstrated the advantage of utilizing two recombinant adenoviral vectors derived from distinct serotypes, Ad type 35 CS (Ad35.CS) and Ad5.CS, in a heterologous prime-boost regimen in mice and nonhuman primates (46). This heterologous prime-boost regimen elicited a high-level CS-specific IFN-γ⁺ T-cell response as well CS-specific Th1-type antibodies able to bind malaria parasites. Though the Ad5-based vectors are very potent vaccines, the high prevalence of preexisting immunity toward Ad5 in the human population hampers their immunogenicity and clinical utility (8, 38). The low seroprevalence of Ad5-neutralizing antibodies in infants of 6 months to 1.5 years of age offers an opportunity to administer Ad5-based vaccines to this population without antibodies interfering and neutralizing the vaccine efficacy (42); however, acceptance of this approach by regulatory agencies may remain difficult to obtain. Novel vaccine vectors based on rare low-seroprevalence Ad serotypes have an advantage of not being hampered by anti-Ad5 immunity while inducing a strong immune response (1, 4, 28, 33, 41).Within this study, we evaluated whether vaccination with Ad35.CS and Ad26.CS can enhance the CS-specific immune response induced by a yeast-produced full-length CS protein vaccine and, in particular, whether the combined vaccination sustainably potentiates the Th1 responses necessary for protection against malaria. The Ad35.CS vaccine candidate is currently being evaluated in a phase 1 clinical study, in partnership with the National Institute of Allergy and Infectious Diseases, and so far, it has been shown to be safe. Candidate Ad35-based vaccines against other infectious diseases, i.e., tuberculosis and HIV infection, have also been clinically evaluated and demonstrated to be safe and immunogenic. Recently, an Ad26 vector vaccine against HIV was also clinically assessed in a phase I study, which showed that a 3-dose regimen of this HIV candidate vaccine is safe and immunogenic. Based upon encouraging results, the clinical testing of the combination of Ad35- and Ad26-based vaccines against malaria and HIV is in preparation.