Immune Signature Against Plasmodium falciparum Antigens Predicts Clinical Immunity in Distinct Malaria Endemic Communities.

A large body of evidence supports the role of antibodies directed against the Plasmodium spp. parasite in the development of naturally acquired immunity to malaria, however an antigen signature capable of predicting protective immunity against Plasmodium remains to be identified. Key challenges for the identification of a predictive immune signature include the high dimensionality of data produced by high-throughput technologies and the limitation of standard statistical tests in accounting for synergetic interactions between immune responses to multiple targets. In this study, using samples collected from young children in Ghana at multiple time points during a longitudinal study, we adapted a predictive modeling framework which combines feature selection and machine learning techniques to identify an antigen signature of clinical immunity to malaria. Our results show that an individual's immune status can be accurately predicted by measuring antibody responses to a small defined set of 15 target antigens. We further demonstrate that the identified immune signature is highly versatile and capable of providing precise and accurate estimates of clinical protection from malaria in an independent geographic community. Our findings pave the way for the development of a robust point-of-care test to identify individuals at high risk of disease and which could be applied to monitor the impact of vaccinations and other interventions. This approach could be also translated to biomarker discovery for other infectious diseases.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.

BACKGROUND: Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development. METHODS: Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model. RESULTS: Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis. CONCLUSIONS: Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.

Novel Plasmodium antigens identified via genome-based antibody screen induce protection associated with polyfunctional T cell responses.

The development of vaccines against complex intracellular pathogens, such as Plasmodium spp., where protection is likely mediated by cellular immune responses, has proven elusive. The availability of whole genome, proteome and transcriptome data has the potential to advance rational vaccine development but yet there are no licensed vaccines against malaria based on antigens identified from genomic data. Here, we show that the Plasmodium yoelii orthologs of four Plasmodium falciparum proteins identified by an antibody-based genome-wide screening strategy induce a high degree of sterile infection-blocking protection against sporozoite challenge in a stringent rodent malaria model. Protection increased in multi-antigen formulations. Importantly, protection was highly correlated with the induction of multifunctional triple-positive T cells expressing high amounts of IFN-γ, IL-2 and TNF. These data demonstrate that antigens identified by serological screening are targets of multifunctional cellular immune responses that correlate with protection. Our results provide experimental validation for the concept of rational vaccine design from genomic sequence data.

Specific humoral response of hosts with variable schistosomiasis susceptibility.

The schistosome blood flukes are some of the largest global causes of parasitic morbidity. Further study of the specific antibody response during schistosomiasis may yield the vaccines and diagnostics needed to combat this disease. Therefore, for the purposes of antigen discovery, sera and antibody-secreting cell (ASC) probes from semi-permissive rats and sera from susceptible mice were used to screen a schistosome protein microarray. Following Schistosoma japonicum infection, rats had reduced pathology, increased antibody responses and broader antigen recognition profiles compared with mice. With successive infections, rat global serological reactivity and the number of recognized antigens increased. The local antibody response in rat skin and lung, measured with ASC probes, increased after parasite migration and contributed antigen-specific antibodies to the multivalent serological response. In addition, the temporal variation of anti-parasite serum antibodies after infection and reinfection followed patterns that appear related to the antigen driving the response. Among the 29 antigens differentially recognized by the infected hosts were numerous known vaccine candidates, drug targets and several S. japonicum homologs of human schistosomiasis resistance markers-the tegument allergen-like proteins. From this set, we prioritized eight proteins that may prove to be novel schistosome vaccine and diagnostic antigens.

Antibody Signatures Reflect Different Disease Pathologies in Patients With Schistosomiasis Due to Schistosoma japonicum.

Infection with Schistosoma japonicum causes high levels of pathology that is predominantly determined by the cellular and humoral response of the host. However, the specific antibody response that arises during the development of disease is largely undescribed in Asian schistosomiasis-endemic populations. A schistosome protein microarray was used to compare the antibody profiles of subjects with acute infection, with early or advanced disease associated with severe pathology, with chronic infection, and subjects exposed but stool negative for S. japonicum eggs to the antibody profiles of nonexposed controls. Twenty-five immunodominant antigens were identified, including vaccine candidates, tetraspanin-related proteins, transporter molecules, and unannotated proteins. Additionally, individuals with severe pathology had a limited specific antibody response, suggesting that individuals with mild disease may use a broad and strong antibody response, particularly against surface-exposed proteins, to control pathology and/or infection. Our study has identified specific antigens that can discriminate between S. japonicum-exposed groups with different pathologies and may also allow the host to control disease pathology and provide resistance to parasite infection.

Probing of a human proteome microarray with a recombinant pathogen protein reveals a novel mechanism by which hookworms suppress B-cell receptor signaling.

Na-ASP-2 is an efficacious hookworm vaccine antigen. However, despite elucidation of its crystal structure and studies addressing its immunobiology, the function of Na-ASP-2 has remained elusive. We probed a 9000-protein human proteome microarray with Na-ASP-2 and showed binding to CD79A, a component of the B-cell antigen receptor complex. Na-ASP-2 bound to human B lymphocytes ex vivo and downregulated the transcription of approximately 1000 B-cell messenger RNAs (mRNAs), while only approximately 100 mRNAs were upregulated, compared with control-treated cells. The expression of a range of molecules was affected by Na-ASP-2, including factors involved in leukocyte transendothelial migration pathways and the B-cell signaling receptor pathway. Of note was the downregulated transcription of lyn and pi3k, molecules that are known to interact with CD79A and control B-cell receptor signaling processes. Together, these results highlight a previously unknown interaction between a hookworm-secreted protein and B cells, which has implications for helminth-driven immunomodulation and vaccine development. Further, the novel use of human protein microarrays to identify host-pathogen interactions, coupled with ex vivo binding studies and subsequent analyses of global gene expression in human host cells, demonstrates a new pipeline by which to explore the molecular basis of infectious diseases.

Protein microarrays for parasite antigen discovery.

The host serological profile to a parasitic infection, such as schistosomiasis, can be used to define potential vaccine and diagnostic targets. Determining the host antibody response using traditional approaches is hindered by the large number of putative antigens in any parasite proteome. Parasite protein microarrays offer the potential for a high-throughput host antibody screen to simplify this task. In order to construct the array, parasite proteins are selected from available genomic sequence and protein databases using bioinformatic tools. Selected open reading frames are PCR amplified, incorporated into a vector for cell-free protein expression, and printed robotically onto glass slides. The protein microarrays can be probed with antisera from infected/immune animals or humans and the antibody reactivity measured with fluorophore labeled antibodies on a confocal laser microarray scanner to identify potential targets for diagnosis or therapeutic or prophylactic intervention.

An immunomics approach to schistosome antigen discovery: antibody signatures of naturally resistant and chronically infected individuals from endemic areas.

Schistosomiasis is a neglected tropical disease that is responsible for almost 300,000 deaths annually. Mass drug administration (MDA) is used worldwide for the control of schistosomiasis, but chemotherapy fails to prevent reinfection with schistosomes, so MDA alone is not sufficient to eliminate the disease, and a prophylactic vaccine is required. Herein, we take advantage of recent advances in systems biology and longitudinal studies in schistosomiasis endemic areas in Brazil to pilot an immunomics approach to the discovery of schistosomiasis vaccine antigens. We selected mostly surface-derived proteins, produced them using an in vitro rapid translation system and then printed them to generate the first protein microarray for a multi-cellular pathogen. Using well-established Brazilian cohorts of putatively resistant (PR) and chronically infected (CI) individuals stratified by the intensity of their S. mansoni infection, we probed arrays for IgG subclass and IgE responses to these antigens to detect antibody signatures that were reflective of protective vs. non-protective immune responses. Moreover, probing for IgE responses allowed us to identify antigens that might induce potentially deleterious hypersensitivity responses if used as subunit vaccines in endemic populations. Using multi-dimensional cluster analysis we showed that PR individuals mounted a distinct and robust IgG1 response to a small set of newly discovered and well-characterized surface (tegument) antigens in contrast to CI individuals who mounted strong IgE and IgG4 responses to many antigens. Herein, we show the utility of a vaccinomics approach that profiles antibody responses of resistant individuals in a high-throughput multiplex approach for the identification of several potentially protective and safe schistosomiasis vaccine antigens.

Genome- and proteome-wide screening strategies for antigen discovery and immunogen design.

Infectious diseases remain a leading global cause of morbidity and mortality and there is an urgent need for effective approaches to develop vaccines, especially against complex pathogens. The availability of comprehensive genomic, proteomic and transcriptomic datasets has shifted the paradigm of vaccine development from microbiological to sequence-based approaches. However, how to effectively translate raw data into candidate vaccines is not yet obvious. Herein, we review cutting-edge technologies and screening strategies to mine genomic sequence information for state-of-the-art rational vaccine design, and highlight recent trends. Interdisciplinary approaches which cross the traditional boundaries of genomics, molecular biology, cell biology, immunology and computer science, and which prioritise antigens according to clinically relevant criteria, offer potential solutions to the widespread threat that complex pathogens pose to public health.

A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics.

Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.

Immunization with apical membrane antigen 1 confers sterile infection-blocking immunity against Plasmodium sporozoite challenge in a rodent model.

Apical membrane antigen 1 (AMA-1) is a leading blood-stage malaria vaccine candidate. Consistent with a key role in erythrocytic invasion, AMA-1-specific antibodies have been implicated in AMA-1-induced protective immunity. AMA-1 is also expressed in sporozoites and in mature liver schizonts where it may be a target of protective cell-mediated immunity. Here, we demonstrate for the first time that immunization with AMA-1 can induce sterile infection-blocking immunity against Plasmodium sporozoite challenge in 80% of immunized mice. Significantly higher levels of gamma interferon (IFN-γ)/interleukin-2 (IL-2)/tumor necrosis factor (TNF) multifunctional T cells were noted in immunized mice than in control mice. We also report the first identification of minimal CD8(+) and CD4(+) T cell epitopes on Plasmodium yoelii AMA-1. These data establish AMA-1 as a target of both preerythrocytic- and erythrocytic-stage protective immune responses and validate vaccine approaches designed to induce both cellular and humoral immunity.

The stability and complexity of antibody responses to the major surface antigen of Plasmodium falciparum are associated with age in a malaria endemic area.

Individuals that are exposed to malaria eventually develop immunity to the disease with one possible mechanism being the gradual acquisition of antibodies to the range of parasite variant surface antigens in their local area. Major antibody targets include the large and highly polymorphic Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of proteins. Here, we use a protein microarray containing 123 recombinant PfEMP1-DBLα domains (VAR) from Papua New Guinea to seroprofile 38 nonimmune children (<4 years) and 29 hyperimmune adults (≥15 years) from the same local area. The overall magnitude, prevalence and breadth of antibody response to VAR was limited at <2 years and 2-2.9 years, peaked at 3-4 years and decreased for adults compared with the oldest children. An increasing proportion of individuals recognized large numbers of VAR proteins (>20) with age, consistent with the breadth of response stabilizing with age. In addition, the antibody response was limited in uninfected children compared with infected children but was similar in adults irrespective of infection status. Analysis of the variant-specific response confirmed that the antibody signature expands with age and infection. This also revealed that the antibody signatures of the youngest children overlapped substantially, suggesting that they are exposed to the same subset of PfEMP1 variants. VAR proteins were either seroprevalent from early in life, (<3 years), from later in childhood (≥3 years) or rarely recognized. Group 2 VAR proteins (Cys2/MFK-REY+) were serodominant in infants (<1-year-old) and all other sequence subgroups became more seroprevalent with age. The results confirm that the anti-PfEMP1-DBLα antibody responses increase in magnitude and prevalence with age and further demonstrate that they increase in stability and complexity. The protein microarray approach provides a unique platform to rapidly profile variant-specific antibodies to malaria and suggests novel insights into the acquisition of immunity to malaria.

Sterile protective immunity to malaria is associated with a panel of novel P. falciparum antigens.

The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized.

TLR9-independent effects of inhibitory oligonucleotides on macrophage responses to S. typhimurium.

Detection of bacterial CpG-containing DNA (CpG DNA) by innate immune cells is dependent on toll-like receptor 9 (TLR9). Here we show that the expression of tlr9 mRNA was induced in mouse bone marrow-derived macrophages (BMMs) upon infection with the facultative Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium (S. typhimurium). Treatment of BMM with the inhibitory oligonucleotide (ODN) 2114, an antagonist of TLR9 signalling, enhanced intracellular S. typhimurium numbers approximately fivefold, whereas a control ODN (2310) had no significant effect. Surprisingly, 2114 also amplified S. typhimurium bacterial loads in TLR9-deficient BMM. Indeed, 2114 suppressed responses (nuclear factor-kappaB-dependent reporter gene expression and interleukin-12p40 secretion) to not only CpG DNA, but also the TLR2 ligand Pam(3)Cys, in BMM and RAW264 cells in a sequence-specific manner. Inhibitory ODNs, which have been proposed as therapeutic agents for the treatment of systemic lupus erythematosus because of their inhibitory effects on TLR9 signalling, may thus compromise the host response to bacterial pathogens through TLR9-independent mechanisms.

PU.1 and ICSBP control constitutive and IFN-gamma-regulated Tlr9 gene expression in mouse macrophages.

Macrophages are activated by unmethylated CpG-containing DNA (CpG DNA) via TLR9. IFN-gamma and LPS can synergize with CpG DNA to enhance proinflammatory responses in murine macrophages. Here, we show that LPS and IFN-gamma up-regulated Tlr9 mRNA in murine bone marrow-derived macrophages (BMM). The ability of LPS and IFN-gamma to induce Tlr9 mRNA expression in BMM was dependent on the presence of the growth factor, CSF-1, which is constitutively present in vivo. However, there were clear differences in mechanisms of Tlr9 mRNA induction. LPS stimulation rapidly removed the CSF-1 receptor (CSF-1R) from the cell surface, thereby blocking CSF-1-mediated transcriptional repression and indirectly inducing Tlr9 mRNA expression. By contrast, IFN-gamma activated the Tlr9 promoter directly and only marginally affected cell surface CSF-1R expression. An approximately 100-bp proximal promoter of the murine Tlr9 gene was sufficient to confer basal and IFN-gamma-inducible expression in RAW264.7 cells. A composite IFN regulatory factor (IRF)/PU.1 site upon the major transcription start site was identified. Mutation of the binding sites for PU.1 or IRF impaired basal promoter activity, but only the IRF-binding site was required for IFN-gamma induction. The mRNA expression of the IRF family member IFN consensus-binding protein [(ICSBP)/IRF8] was coregulated with Tlr9 in macrophages, and constitutive and IFN-gamma-inducible Tlr9 mRNA expression was reduced in ICSBP-deficient BMM. This study therefore characterizes the regulation of mouse Tlr9 expression and defines a molecular mechanism by which IFN-gamma amplifies mouse macrophage responses to CpG DNA.

CpG DNA activates survival in murine macrophages through TLR9 and the phosphatidylinositol 3-kinase-Akt pathway.

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-alpha production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression.

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

DNA motifs suppressing TLR9 responses.

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythematosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleotides (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action.

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor (tlr)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R.