Identification of a neutralizing epitope within minor repeat region of Plasmodium falciparum CS protein

Malaria remains a major cause of morbidity and mortality worldwide with 219 million infections and 435,000 deaths predominantly in Africa. The infective Plasmodium sporozoite is the target of a potent humoral immune response that can protect murine, simian and human hosts against challenge by malaria-infected mosquitoes. Early murine studies demonstrated that sporozoites or subunit vaccines based on the sporozoite major surface antigen, the circumsporozoite (CS) protein, elicit antibodies that primarily target the central repeat region of the CS protein. In the current murine studies, using monoclonal antibodies and polyclonal sera obtained following immunization with P. falciparum sporozoites or synthetic repeat peptides, we demonstrate differences in the ability of these antibodies to recognize the major and minor repeats contained in the central repeat region. The biological relevance of these differences in fine specificity was explored using a transgenic P. berghei rodent parasite expressing the P. falciparum CS repeat region. In these in vitro and in vivo studies, we demonstrate that the minor repeat region, comprised of three copies of alternating NANP and NVDP tetramer repeats, contains an epitope recognized by sporozoite-neutralizing antibodies. In contrast, murine monoclonal antibodies specific for the major CS repeats (NANP)n could be isolated from peptide-immunized mice that had limited or no sporozoite-neutralizing activity. These studies highlight the importance of assessing the fine specificity and functions of antirepeat antibodies elicited by P. falciparum CS-based vaccines and suggest that the design of immunogens to increase antibody responses to minor CS repeats may enhance vaccine efficacy

Human CD4+ T Cell Epitopes from Vaccinia Virus Induced by Vaccination or Infection

Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4+ T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4+ T cell responses have been poorly characterized, and CD4+ T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens

Phase I Trial of an Alhydrogel Adjuvanted Hepatitis B Core Virus-Like Particle Containing Epitopes of Plasmodium falciparum Circumsporozoite Protein

The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide) in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax) is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from Plasmodium falciparum circumsporozoite protein containing an immunodominant B [(NANP)3] epitope, an HLA-restricted CD4 (NANPNVDPNANP) epitope and a universal T cell epitope (T*) (amino acids 326—345, NF54 isolate). We assessed an Alhydrogel (aluminum hydroxide)-adjuvanted vaccine formulation at three ICC-1132 dose levels, each injected intramuscularly (1.0 mL) on study days 0, 56 and 168. A saline vaccine formulation was found to be unstable after prolonged storage and this formulation was subsequently removed from the study. Thirty-two volunteers were followed for one year. Local and systemic adverse clinical events were measured and immune responses to P. falciparum and hepatitis B virus core antigens were determined utilizing the following assays: IgG and IgM ELISA, indirect immunofluorescence against P. falciparum sporozoites, circumsporozoite precipitin (CSP) and transgenic sporozoite neutralization assays. Cellular responses were measured by proliferation and IL-2 assays. Local and systemic reactions were similarly mild and well tolerated between dose cohorts. Depending on the ICC-1132 vaccine concentration, 95 to 100% of volunteers developed antibody responses to the ICC-1132 immunogen and HBc after two injections; however, only 29—75% and 29—63% of volunteers, respectively, developed malaria-specific responses measured by the malaria repeat synthetic peptide ELISA and IFA; 2 of 8 volunteers had positive reactions in the CSP assay. Maximal transgenic sporozoite neutralization assay inhibition was 54%. Forty-seven to seventy-five percent demonstrated T cell proliferation in response to ICC-1132 or to recombinant circumsporozoite protein (rCS) NF-54 isolate. This candidate malaria vaccine was well tolerated, but the vaccine formulation was poorly immunogenic. The vaccine may benefit from a more powerful adjuvant to improve immunogenicity

An unstable Th epitope of P. falciparum fosters central memory T cells and anti-CS antibody responses

Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRbeta1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-gamma responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed

Why Functional Pre-Erythrocytic and Bloodstage Malaria Vaccines Fail: A Meta-Analysis of Fully Protective Immunizations and Novel Immunological Model

Background: Clinically protective malaria vaccines consistently fail to protect adults and children in endemic settings, and at best only partially protect infants. Methodology/Principal Findings: We identify and evaluate 1916 immunization studies between 1965-February 2010, and exclude partially or nonprotective results to find 177 completely protective immunization experiments. Detailed reexamination reveals an unexpectedly mundane basis for selective vaccine failure: live malaria parasites in the skin inhibit vaccine function. We next show published molecular and cellular data support a testable, novel model where parasite-host interactions in the skin induce malaria-specific regulatory T cells, and subvert early antigen-specific immunity to parasite-specific immunotolerance. This ensures infection and tolerance to reinfection. Exposure to Plasmodium-infected mosquito bites therefore systematically triggers immunosuppression of endemic vaccine-elicited responses. The extensive vaccine trial data solidly substantiate this model experimentally. Conclusions/Significance: We conclude skinstage-initiated immunosuppression, unassociated with bloodstage parasites, systematically blocks vaccine function in the field. Our model exposes novel molecular and procedural strategies to significantly and quickly increase protective efficacy in both pipeline and currently ineffective malaria vaccines, and forces fundamental reassessment of central precepts determining vaccine development. This has major implications fo

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B

Ex-vivo IFN-γ production by PBMCs from healthy donors in response to HHV-6B antigens (extracellular virus pellet and infected-cell lysate).

<p>A. Total IFN-γ in cell culture supernatant measured by ELISA (fg/cell) after 120 hours incubation of PBMCs with antigens. B. Total number of IFN-γ-producing cells measured by ELISpot (SFU/10⁶ cells) after 48 hours incubation of PBMCs with antigens. C. Effect of depletion of CD4, CD8 or CD19 subpopulations on IFN-γ production by total PBMCs, measured by ELISpot (presented as percentage of the response of total PBMCs). Statistical analysis: parametric paired t-test. Each donor is represented by the same colored symbol in the different graphs.</p

Validation of the IFN-γ response to selected HHV-6B peptides.

<p>Peptide-expanded T cell cultures to each of the 14 peptides in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142871#pone.0142871.t004" target="_blank">Table 4</a> were generated for 3 donors, and tested for IFN-γ using ELISpot (SFU/10⁶ cells). A. Responses of all expanded T cells to autologous PBMCs pulsed with the peptide (P) or HHV-6B (V). ELISpot negative controls are indicated by (-). Positive responses were assessed by DFR2x and are indicated by (+) on top of the bar. B. Summary of the responses of expanded T cells to autologous PBMCs pulsed with HCMV or HHV-6B for two HCMV seronegative (#118 and 132) and one seropositive (#131) donor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142871#pone.0142871.t001" target="_blank">Table 1</a>). P-values for HCMV vs HHV-6B (unpaired t-test) are shown. C. Alignment of homologous HHV-6B U57 and HCMV UL86 sequences in the region of known epitopes (boxes indicate the complete stimulating sequence). D. Response of a 6BZ_1084 CD4 T cell clone to autologous BLCLs or PBMCs pulsed with HHV-6B 6BZ_1084 or with HCMV UL86-B peptides, as measured by IFN-γ ELISA. P-values for no-peptide vs peptide (unpaired t-test) are shown.</p

Mapping of the CD4 T cell responses in PBMCs to HHV-6B proteins.

<p>A. IFN-γ ELISpot responses (SFU/10⁶ cells) by donors #037, 118 and 132 to pools 1–29. Error bars represent the standard deviation of the replicates. B. Heat maps summarizing the response of all donors (n = 5) to all pools tested (53). Positive responses were assessed by DFR2x and ER analyses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142871#sec002" target="_blank">Methods</a>). Pools for which positive responses were observed by DFR2x in at least 4 donors were selected for further analysis and are indicated by (+). C. IFN-γ ELISpot responses by different donors to individual peptides in pools 5, 11, 23 and 29. D. Heat-maps summarizing the IFN-γ responses to individual peptides present in the pools selected in B. Positive responses to individual peptides were assessed by ER and DFR2x analyses. Fourteen peptides that induced responses in multiple donors were selected for further validation, and are indicated by (+).</p

Identification of HHV-6B targets of the IFN-γ response.

<p>A. Schematic of the experimental approach: fractionation of proteins by SDS-PAGE; measurement of IFN-γ secretion induced by gel fractions; identification of HHV-6B proteins by proteomics in selected fractions. B and C. IFN-γ production by PBMCs (B) or in vitro HHV-6B-expanded T cell cultures (C), in response to stimulation with gel fractions containing HHV-6B proteins. Middle panels show SDS-PAGE profiles of extracellular virus; left panels show IFN-γ production (fg/cell) by each fraction; right panels show protein identification by proteomics (pie charts show the numbers of tryptic peptides characterized by mass spectrometry used to identify each HHV-6B protein; numbers are shown in parentheses). In B, individual IFN-γ responses of two different donors are shown using black (#118) and grey (#131) bars, with filled bars indicating positive responses (values above the background mean + 3 times standard deviation). Error bars represent standard deviation of replicate measurements for each donor. Filled-bars indicate fractions selected for proteomics analysis. In C, average IFN-γ responses of four expanded T cell cultures (from two different donors); error bars represent the standard deviation. Filled bars indicate positive responses (values above the background mean + 3 times the standard deviation), selected for proteomics analysis.</p