Occurrence of the human tumor-specific antigen structure Galβ1-3GalNAcα- (Thomsen-Friedenreich) and related structures on gut bacteria: prevalence, immunochemical analysis and structural confirmation

The Thomsen-Friedenreich antigen (TF; CD176, Galβ1-3GalNAcα-) is a tumor-specific carbohydrate antigen and a promising therapeutic target. Antibodies that react with this antigen are frequently found in the sera of healthy adults and are assumed to play a role in cancer immunosurveillance. In this study, we examined the occurrence of α-anomeric TF (TFα) on a large variety of gastrointestinal bacteria using a novel panel of well-characterized monoclonal antibodies. Reactivity with at least one anti-TF antibody was found in 13% (16 of 122) of strains analyzed. A more in-depth analysis, using monoclonal antibodies specific for α- and β-anomeric TF in combination with periodate oxidation, revealed that only two novel Bacteroides ovatus strains (D-6 and F-1), isolated from the faeces of healthy persons by TF-immunoaffinity enrichment, possessed structures that are immunochemically identical to the true TFα antigen. The TF-positive capsular polysaccharide structure of strain D-6 was characterized by mass spectrometry, monosaccharide composition analysis, glycosidase treatments and immunoblot staining with TFα- and TFβ-specific antibodies. The active antigen was identified as Galβ1-3GalNAc-, which was α-anomerically linked as a branching structure within a heptasaccharide repeating unit. We conclude that structures immunochemically identical to TFα are extremely rare on the surface of human intestinal bacteria and may only be identifiable by binding of both antibodies, NM-TF1 and NM-TF2, which recognize a complete immunomolecular imprint of the TFα structure. The two novel B. ovatus strains isolated in this study may provide a basis for the development of TF-based anti-tumor vaccines.     

Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-α

The focus of our work on rapid actions of estrogens has been on the immuno-identification of a membrane version of the estrogen receptor-α (mERα) and the correlation of the presence of this receptor to the rapid secretion of prolactin in pituitary tumor cells. We demonstrated the mERα by both fluorescence and immuno-enzyme-cytochemistry and with both conventional and confocal microscopy in the cell line GH₃/B6 and its sublines. Its presence on cells (including recently subcloned ones) is very heterogenous, unlike the nuclear ERα, which is present in every cell. An impeded ligand (estradiol covalently linked to BSA) binds to mERα and elicits the same response. A total of eight antibodies to ERα recognize mERα, making it likely that the membrane and nuclear proteins are highly related. Immuno-identification techniques have also been used to identify mERα on the MCF-7 human breast cancer cell line. Estradiol at very low concentrations elicits prolactin release from GH₃/B6 cells within a few minutes of application. This response is bimodal, with effective concentrations in both the picomolar and nanomolar ranges. Prolactin release is also elicited or inhibited by ERα-specific antibodies. The characteristics of mERα and the membrane receptor for glucocorticoids have many similarities, suggesting that this mode of subcellular location/function alternative might be used by other members of the gene family.     

Naturally acquired antibodies to Plasmodium vivax blood-stage vaccine candidates (PvMSP-1₁₉ and PvMSP-3α₃₅₉₋₇₉₈ and their relationship with hematological features in malaria patients from the Brazilian Amazon

An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-1₁₉ and PvMSP-3α_359?798. Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-1₁₉ was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3α_359?798. Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3α), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3α_359?798 during natural infection.     

Preliminary results in HIV-1-infected patients treated with transfer factor (TF) and Zidovudine (ZDV)

The efficiency of HIV-1 specific transfer factor (TF) administration, combined with Zidovudine (ZDV), in asymptomatic persistent generalised lymphadenopaty, or AIDS related complex (ARC) patients was evaluated. Twenty patients were randomly assigned to receive only ZDV (1st group) or ZDV together with HIV-1-specific TF (2nd group). HIV-1-specific TF was administered orally at 2 × 10⁷ cell equivalent daily for 15 days, and thereafter once a week for up to 6 months. There were no significant differences between the two groups in clinical evolution, red blood cells, haemoglobin, lymphocytes, CD20 subset, transaminases,β-2-microglobulin, p24 antigen. White blood cells, CD8 lymphocytes as well as IL-2 levels increased in the second group, while the CD4 subset increased in the first group. The combination treatment with ZDV and TF appeared to be safe and well tolerated. Furthermore, levels of serum cytokines were investigated in 10 patients (8 asymptomatic and 2 ARC) treated with ZDV, and compared with 5 patients of the 2nd group (3 asymptomatic and 2 ARC) treated with ZDV plus HIV-1-specific TF. Peripheral lymphocytes, CD4, CD8 subsets, IL-2, TNFα, IL-6, p24 antigen, IL-2 soluble lymphocyte receptors (sR), CD4sR, CD8sR and ß-2-microglobulin were evaluated at the baseline and at the 3rd month. The CD4 subset was not significantly different in the two groups, whilst IL-2 increased in the 2nd group receiving ZDV plus TF, suggesting an activation of the Th1 secretion pattern.     

Deletion of Fifteen Open Reading Frames from Modified Vaccinia Virus Ankara Fails to Improve Immunogenicity

Modified vaccinia virus Ankara (MVA) is a highly attenuated strain of vaccinia virus, which has been used as a recombinant vaccine vector in many vaccine development programmes. The loss of many immunosuppressive and host-range genes resulted in a safe and immunogenic vaccine vector. However it still retains some immunomodulatory genes that may reduce MVA immunogenicity. Earlier reports demonstrated that the deletion of the A41L, B15R, C6L, or C12L open reading frames (ORFs) enhanced cellular immune responses in recombinant MVA (rMVA) by up to 2-fold. However, previously, we showed that deletion of the C12L, A44L, A46R, B7R, or B15R ORFs from rMVA, using MVA-BAC recombineering technology, did not enhance rMVA immunogenicity at either peak or memory cellular immune responses. Here, we extend our previous study to examine the effect of deleting clusters of genes on rMVA cellular immunogenicity. Two clusters of fifteen genes were deleted in one rMVA mutant that encodes either the 85A antigen of Mycobacterium tuberculosis or an immunodominant H2-K^d-restricted murine malaria epitope (pb9). The deletion mutants were tested in prime only or prime and boost vaccination regimens. The responses showed no improved peak or memory CD8⁺ T cell frequencies. Our results suggest that the reported small increases in MVA deletion mutants could not be replicated with different antigens, or epitopes. Therefore, the gene deletion strategy may not be taken as a generic approach for improving the immunogenicity of MVA-based vaccines, and should be carefully assessed for every individual recombinant antigen.     

Immune Responses to HBsAg Conjugated to Protein D of Non-Typeable Haemophilus influenzae in Mice

BackgroundHepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1–6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten–carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine.ConclusionsRecombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines.     

Immunological features and efficacy of the reconstructed epitope vaccine CtUBE against Helicobacter pylori infection in BALB/c mice model

Urease is an essential virulence factor and colonization factor for Helicobacter pylori, of which the urease B subunit (UreB) is considered as an excellent vaccine candidate antigen. In previous study, an epitope vaccine with cholera toxin B subunit (CTB) and an epitope (UreB_321–339) named CtUBE was constructed and the mice were protected significantly after intragastric vaccination with the CtUBE liposome vaccine. However, the fusion protein CtUBE was expressed as inclusion bodies and was difficultly purified. Besides, the immunogenicity and specificity of the CtUBE vaccine was not investigated in a fairly wide and detailed way. In this study, the fusion peptide CtUBE was reconstructed and expressed as a soluble protein with pectinase signal peptide at the N terminus and the 6-his tag at its C-terminal, and then the immunogenicity, specificity, prophylactic, and therapeutic efficacy of the reconstructed CtUBE (rCtUBE) vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that mice immunized with rCtUBE could produce comparatively high level of specific antibodies which could respond to natural H. pylori urease, UreB, or the minimal epitope UreB_327–334 involved with the active site of urease, and showed effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with rCtUBE significantly decreased H. pylori colonization compared with oral immunization with rCTB or PBS, and the protection was correlated with antigen-specific IgG, IgA, and mucosal sIgA antibody responses, and a Th2 cells response. This rCtUBE vaccine may be a promising vaccine candidate for the control of H. pylori infection.     

First Detection and Molecular Identification of Borrelia garinii Spirochete from Ixodes ovatus Tick Ectoparasitized on Stray Cat in Taiwan

Borrelia garinii spirochete was detected for the first time in Ixodes ovatus tick ectoparasitized on stray cat in Taiwan. The genetic identity of this detected spirochete was determined by analyzing the gene sequence amplified by genospecies-specific polymerase chain reaction assays based on the 5S–23S intergenic spacer amplicon (rrf-rrl) and outer surface protein A (ospA) genes of B. burgdorferi sensu lato. Phylogenetic relationships were analyzed by comparing the sequences of rrf-rrl and ospA genes obtained from 27 strains of Borrelia spirochetes representing six genospecies of Borrelia. Seven major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. Phylogenetic analysis based on rrf-rrl gene revealed that this detected spirochete (strain IO-TP-TW) was genetically affiliated to the same clade with a high homogeneous sequences (96.7 to 98.1% similarity) within the genospecies of B. garinii and can be discriminated from other genospecies of Borrelia spirochetes. Interspecies analysis based on the genetic distance values indicates a lower level (<0.022) of genetic divergence (GD) within the genospecies of B. garinii, and strain IO-TP-TW was genetically more distant ( >0.113) to the strains identified in I. ovatus collected from Japan and China. Intraspecies analysis also reveals a higher homogeneity (GD<0.005) between tick (strain IO-TP-TW) and human (strain Bg-PP-TW1) isolates of B. garinii in Taiwan. This study provides the first evidence of B. garinii isolated and identified in an I. ovatus tick in Asia, and the higher homogeneity of B. garinii between tick and human strain may imply the risk of human infection by I. ovatus bite.     

Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.     

In vitro antibody response to tetanus in the MIMIC™ system is a representative measure of vaccine immunogenicity

In response to the recurrent failure of animal vaccine protection studies to accurately predict human trial results, we have developed a fully human modular immune in vitro construct (MIMIC?) to serve as a preliminary screen for efficacy testing of potential vaccine formulations. To validate the potential of this approach, we monitored the in vitro-generated tetanus (TT)-specific antibody levels in a cohort of donors before and after receiving tetanus vaccination. Purified CD4_T cell and B cell populations were combined with autologous tetanus vaccine-pulsed dendritic cells to generate specific antibody. Enumeration of TT-specific IgG antibody-secreting cells by ELISPOT displayed a significant increase in the magnitude of this population after vaccination. The relative magnitudes of the in vitro-generated TT-specific antibody response before and after vaccination largely recapitulated the TT-specific IgG serum titer profiles measured in the same individuals. These findings provide evidence that the MIMIC? system can be a rapid and representative in vitro method for measuring vaccine immunogenicity via induction of the memory B cell response.     

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFNγ and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.     

A Vaccine Targeted at CETP Alleviates High Fat and High Cholesterol Diet-Induced Atherosclerosis and Non-Alcoholic Steatohepatitis in Rabbit

Low HDL-C levels are associated with atherosclerosis and non-alcoholic steatohepatitis, and increased levels may reduce the risk of these diseases. Inhibition of cholesteryl ester transfer protein (CETP) activity is considered a promising strategy for increasing HDL-C levels. Since CETP is a self-antigen with low immunogenicity, we developed a novel CETP vaccine (Fc-CETP₆) to overcome the low immunogenicity of CETP and for long-term inhibition of CETP activity. The vaccine consists of a rabbit IgG Fc domain for antigen delivery to antigen-presenting cells fused to a linear array of 6 repeats of a CETP epitope to efficiently activate B cells. Rabbits were fed a high fat/cholesterol (HFC) diet to induce atherosclerosis and NASH, and immunized with Fc-CETP₆ vaccine. The Fc-CETP₆ vaccine successfully elicited anti-CETP antibodies and lowered plasma CETP activity. The levels of plasma HDL-C and ApoA-I were higher, and plasma ox-LDL lower, in the Fc-CETP₆-immunized rabbits as compared to the unimmunized HFC diet-fed rabbits. Pathological analyses revealed less lipid accumulation and inflammation in the aorta and liver of the Fc-CETP₆-immunized rabbits. These results show that the Fc-CETP₆ vaccine efficiently elicited antibodies against CETP and reduced susceptibility to both atherosclerosis and steatohepatitis induced by the HFC diet. Our findings suggest that the Fc-CETP₆ vaccine may improve atherosclerosis and NASH and has high potential for clinical use.     

Exosomes derived from tumor cells genetically modified to express <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigen: a novel vaccine for cancer therapy

Objectives

To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens.

Results

We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p < 0.05) evoked cellular immunity against both ESAT-6, and B16 tumor cells. Intra-tumoral injection of the exosomes significantly suppressed (p < 0.001) tumor growth in syngeneic B16 tumor-bearing mice, while the exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens.

Conclusions

Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

     

Brucella abortus Strain RB51 Leucine Auxotroph as an Environmentally Safe Vaccine for Plasmid Maintenance and Antigen Overexpression

To avoid potentiating the spread of an antibiotic resistance marker, a plasmid expressing a leuB gene and a heterologous antigen, green fluorescent protein (GFP), was shown to complement a leucine auxotroph of cattle vaccine strain Brucella abortus RB51, which protected CD1 mice from virulent B. abortus 2308 and elicited GFP antibodies.     

Expression, purification, and immunogenic characterization of Epstein–Barr virus recombinant EBNA1 protein in Pichia pastoris

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390–641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4⁺ and CD8⁺ T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.     

Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies.     

Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1?2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.     

The Effect of Human Factor H on Immunogenicity of Meningococcal Native Outer Membrane Vesicle Vaccines with Over-Expressed Factor H Binding Protein

The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH), is fH-binding protein (fHbp), which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV) from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (P = 0.001) and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (P = 0.003). By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (P = 0.002), and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (P = 0.001). Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans.