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Seasonal “common-cold” human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but identification and characterization of the T cell response to seasonal human coronaviruses remain largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal human coronavirus OC43. We identified MHC-I and MHC-II bound peptides derived from the viral spike, nucleocapsid, hemagglutinin-esterase, 3C-like proteinase, and envelope proteins. Only three MHC-I bound OC43-derived peptides were observed, possibly due to the potent MHC-I downregulation induced by OC43 infection. By contrast, 80 MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. These peptides elicited low-abundance recall T cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T cell lines. Among the validated epitopes, S903-917presented by DPA1*01:03/DPB1*04:01 and S1085-1099presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. N54-68and HE128-142presented by DRB1*15:01 and HE259-273presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow T cell cross-reactivity after infection or vaccination and could aid in the selection of epitopes for inclusion in pan-coronavirus vaccines.Author SummaryThere is much current interest in cellular immune responses to seasonal common-cold coronaviruses because of their possible role in mediating protection against SARS-CoV-2 infection or pathology. However, identification of relevant T cell epitopes and systematic studies of the T cell responses responding to these viruses are scarce. We conducted a study to identify naturally processed and presented MHC-I and MHC-II epitopes from human cells infected with the seasonal coronavirus HCoV-OC43, and to characterize the T cell responses associated with these epitopes. We found epitopes specific to the seasonal coronaviruses, as well as epitopes cross-reactive between HCoV-OC43 and SARS-CoV-2. These epitopes should be useful in following immune responses to seasonal coronaviruses and identifying their roles in COVID-19 vaccination, infection, and pathogenesis.

Background Evaluating the performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and clearly articulating the utility of selected antigens, isotypes, and thresholds is crucial to understanding the prevalence of infection within selected communities. Methods This cross-sectional study, implemented in 2020, screened PCR–confirmed coronavirus disease 2019 patients (n = 86), banked prepandemic and negative samples (n = 96), healthcare workers and family members (n = 552), and university employees (n = 327) for anti–SARS-CoV-2 receptor-binding domain, trimeric spike protein, and nucleocapsid protein immunoglobulin (Ig)G and IgA antibodies with a laboratory-developed enzyme-linked immunosorbent assay and tested how antigen, isotype and threshold choices affected the seroprevalence outcomes. The following threshold methods were evaluated: (i) mean + 3 standard deviations of the negative controls; (ii) 100% specificity for each antigen-isotype combination; and (iii) the maximal Youden index. Results We found vastly different seroprevalence estimates depending on selected antigens and isotypes and the applied threshold method, ranging from 0.0% to 85.4%. Subsequently, we maximized specificity and reported a seroprevalence, based on more than one antigen, ranging from 9.3% to 25.9%. Conclusions This study revealed the importance of evaluating serosurvey tools for antigen-, isotype-, and threshold-specific sensitivity and specificity, to interpret qualitative serosurvey outcomes reliably and consistently across studies.

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4 + immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.

Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S

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.

Understanding the targets of adaptive immunity to SARS-CoV-2 is essential for vaccine development and interpretation of coronavirus disease 2019 (COVID-19) pathogenesis. SARS-CoV-2 and other human coronaviruses share substantial sequence homology raising the possibility that the previous exposure to seasonal “common cold” human coronaviruses could impact the T cell response upon SARS-CoV-2 infection. Detailed investigation of the antigenic peptides presented in SARS-CoV-2 and other common coronavirus infections would be beneficial to understand the impact of preexisting T cell mediated immunity on SARS-CoV-2 infection. The repertoire of naturally processed and presented viral peptides by MHC II upon SARS-CoV-2 and seasonal human coronaviruses infections remains largely uncharacterized. Here, we report the MHC I and II immunopeptidome of cells infected with SARS-CoV-2 or hCoV-OC43, one of the seasonal coronaviruses. We identified 11 MHC I peptides and 13 MHC II peptides of SARS-CoV-2 infected cells including both canonical and out-of-frame spike epitopes, 27 MHC I and 91 MHC II peptides from membrane, nucleocapsid, spike and hemagglutinin-esterase from OC43 infected cells using mass spectrometry. We validated some peptides using HLA peptide binding assays, and these peptides were shown to recall T cell responses in donors with presumed history of common cold viral infection. Some of these peptides share substantial homology with common cold coronaviruses, indicating possibility of conserved T cell epitopes eliciting the protective immunity in COVID-19. The identification of naturally presented peptides and their striking homology with other coronavirus could aid in the peptide selection for the vaccine development.

Plasmodium vivax is the most widespread species of Plasmodium , causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4 + and CD8 + PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development.

Most adults remain chronically infected with HHV-6 after resolution of a primary infection in childhood, with the latent virus held in check by the immune system. Iatrogenic immunosuppression following solid organ transplantation (SOT) or hematopoetic stem cell transplantation (HSCT) can allow latent viruses to reactivate. HHV-6 reactivation has been associated with increased morbidity, graft rejection, and neurological complications post-transplantation. Recent work has identified HHV-6 antigens that are targeted by the CD4+ and CD8+ T cell response in chronically infected adults. T cell populations recognizing these targets can be expanded in vitro and are being developed for use in autologous immunotherapy to control post-transplantation HHV-6 reaction.

Using a consensus epitope prediction approach, three rotavirus (RV) peptides that induce cytokine secretion by CD4 T cells from healthy volunteers were identified. The peptides were shown to bind HLA-DRB1⁎0101 and then used to generate MHC II tetramers. RV specific T cell lines specific for one of the three peptides studied were restricted by MHC class II molecules and contained T cells that bound the tetramer and secreted cytokines upon activation with the peptide. The majority of RV and Flu tetramer+ CD4 T cells in healthy volunteers expressed markers of antigen experienced T cells, but only RV specific CD4 T cells expressed intestinal homing receptors. CD4 T cells from children that received a RV vaccine, but not placebo recipients, were stained with the RV-VP6 tetramer and also expressed intestinal homing receptors. Circulating RV-specific CD4 T cells represent a unique subset that expresses intestinal homing receptors.

Immunization with vaccinia virus elicits a protective Ab response that is almost completely CD4+ T cell dependent. A recent study in a rodent model observed a deterministic linkage between Ab and CD4+ T cell responses to particular vaccinia virus proteins suggesting that CD4+ T cell help is preferentially provided to B cells with the same protein specificity (Sette et al. 2008. Immunity 28: 847–858). However, a causal linkage between Ab and CD4+ T cell responses to vaccinia or any other large pathogen in humans has yet to be done. In this study, we measured the Ab and CD4+ T cell responses against four vaccinia viral proteins (A27L, A33R, B5R, and L1R) known to be strongly targeted by humoral and cellular responses induced by vaccinia virus vaccination in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors. Our data indicate that there is no direct linkage between Ab and CD4+ T cell responses against each individual protein in both short-term and long-term immunized donors. Together with the observation that the presence of immune responses to these four proteins is linked together within donors, our data suggest that in vaccinia-immunized humans, individual viral proteins are not the primary recognition unit of CD4+ T cell help for B cells. Therefore, we have for the first time, to our knowledge, shown evidence that CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to generate robust Ab responses against four vaccinia viral proteins in humans.

The most widespread Plasmodium species, Plasmodium vivax, poses a significant public health threat. An effective vaccine is needed to reduce global malaria burden. Of the erythrocytic stage vaccine candidates, the 19 kDa fragment of the P. vivax Merozoite Surface Protein 1 (PvMSP119) is one of the most promising. Our group has previously defined several promiscuous T helper epitopes within the PvMSP1 protein, with features that allow them to bind multiple MHC class II alleles. We describe here a P. vivax recombinant modular chimera based on MSP1 (PvRMC-MSP1) that includes defined T cell epitopes genetically fused to PvMSP119. This vaccine candidate preserved structural elements of the native PvMSP119 and elicited cytophilic antibody responses and CD4+ and CD8+ T cells capable of recognizing PvMSP119. Although CD8+ T cells that recognize blood stage antigens have been reported to control blood infection, CD8+ T cell responses induced by P. falciparum or P. vivax vaccine candidates based on MSP119 have not been reported. To our knowledge, this is the first time a protein based subunit vaccine has been able to induce CD8+ T cell against PvMSP119. The PvRMC-MSP1 protein was also recognized by naturally acquired antibodies from individuals living in malaria endemic areas with an antibody profile associated with protection from infection. These features make PvRMC-MSP1 a promising vaccine candidate.

HLA-DM (DM) is a nonclassical MHC class II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during Ag presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of DM in epitope selection is still unclear, especially in human response to infectious disease. In this study, we addressed this question in the context of the human CD4 T cell response to vaccinia virus. We measured the IC50, intrinsic dissociation t1/2, and DM-mediated dissociation t1/2 for a large set of peptides derived from the major core protein A10L and other known vaccinia epitopes bound to HLA-DR1 and compared these properties to the presence and magnitude of peptide-specific CD4+ T cell responses. We found that MHC II–peptide complex kinetic stability in the presence of DM distinguishes T cell epitopes from nonrecognized peptides in A10L peptides and also in a set of predicted tight binders from the entire vaccinia genome. Taken together, these analyses demonstrate that DM-mediated dissociation t1/2 is a strong and independent factor governing peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability in the presence of DM.

Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4 + T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro . Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4 + T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4 + T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4 + T cells in immunised volunteers. Both lytic and non-lytic CD4 + T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4 + T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4 + cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4 + CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8 + CTLs, and these multifunctional sporozoite- and peptide-induced CD4 + T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.

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.

An effective malaria vaccine is needed to address the public health tragedy resulting from the high levels of morbidity and mortality caused by Plasmodium parasites. The first protective immune mechanism identified in the irradiated sporozoite vaccine, the “gold standard” for malaria preerythrocytic vaccines, was sporozoite-neutralizing antibody specific for the repeat region of the surface circumsporozoite (CS) protein. Previous phase I studies demonstrated that a branched peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum CS protein elicited antirepeat antibody and CD4 + -T-cell responses comparable to those observed in volunteers immunized with irradiated P. falciparum sporozoites. The current study compares the immunogenicity of linear versus tetrabranched peptides containing the same minimal T- and B-cell epitopes, T1BT*, comprised of a CS-derived universal Th epitope (T*) synthesized in tandem with the T1 and B repeats of P. falciparum CS protein. A simple 48-mer linear synthetic peptide was found to elicit antisporozoite antibody and gamma interferon-secreting T-cell responses comparable to the more complex tetrabranched peptides in inbred strains of mice. The linear peptide was also immunogenic in outbred nonhuman primates ( Aotus nancymaae ), eliciting antibody titers equivalent to those induced by tetrabranched peptides. Importantly, the 48-mer linear peptide administered in adjuvants suitable for human use elicited antibody-mediated protection against challenge with rodent malaria transgenic sporozoites expressing P. falciparum CS repeats. These findings support further evaluation of linear peptides as economical, safe, and readily produced malaria vaccines for the one-third of the world's population at risk of malaria infection.

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.

We report the first phase I trial to assess the safety and immunogenicity of a malaria vaccine candidate, ICC-1132 (Malarivax), composed of a modified hepatitis B virus core protein (HBc) containing minimal epitopes of thePlasmodium falciparumcircumsporozoite (CS) protein. When expressed inEscherichia coli, the recombinant ICC-1132 protein forms virus-like particles that were found to be highly immunogenic in preclinical studies of mice and monkeys. Twenty healthy adult volunteers received a 20- or a 50-μg dose of alum-adsorbed ICC-1132 administered intramuscularly at 0, 2, and 6 months. The majority of volunteers in the group receiving the 50-μg dose developed antibodies to CS repeats as well as to HBc. Malaria-specific T cells that secreted gamma interferon were also detected after a single immunization with ICC-1132-alum. These studies support ICC-1132 as a promising malaria vaccine candidate for further clinical testing using more-potent adjuvant formulations and confirm the potential of modified HBc virus-like particles as a delivery platform for vaccines against other human pathogens.

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-DRβ1*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-γ 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.

Human herpesvirus 6A (HHV-6A) and HHV-6B are betaherpesviruses widely spread in human populations. Both viruses can be differentiated by their biological properties, including host cell, outcome of primary infection, and interactions with the immune system. HHV-6A and HHV-6B are T lymphotropic and neurotropic viruses characterized by immunomodulatory properties mediated by direct effects in infected cells and indirect effects on uninfected cell, including suppression of antigen presentation, cytokine production, and T-cell proliferation. T-cell responses to HHV-6 have been difficult to measure ex vivo in peripheral blood cells, and most studies have characterized T-cell responses after in vitro expansion. These studies have identified primary and secondary polyfunctional CD4+and CD8+T-cell responses. As observed for other large DNA viruses, HHV-6 virion proteins seem to be major targets of the T-cell response. Recent studies have identified several epitopes for CD4 and CD8 T-cell recognition.

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT*)4-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T*, which was originally defined using CD4+ T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T*-specific cellular responses with high anti-repeat Ab titers suggests that the T* epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.

Successful immunity requires that a limited pool of αβ T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by ‘self’ major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA306–318) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA306–318 presented by multiple MHC-II alleles.

Class II MHC proteins bind peptides and present them to CD4 (+) T cells as part of the immune system's surveillance of bodily tissues for foreign and pathogenic material. Antigen processing and presentation pathways have been characterized in detail in normal cells, but there is little known about the actual viral peptides that are presented to CD4 (+) T cells that signal infection. In this study, two-dimensional LC-MS/MS was used to identify vaccinia virus-derived peptides among the hundreds to thousands of peptide antigens bound to the human class II MHC protein HLA-DR1 on the surface of vaccinia virus-infected cells. The peptides, derived from the I6L, D6R, and A10L viral proteins, were 15 residues in length, bound efficiently to HLA-DR1 as synthetic peptides, and were recognized by vaccinia-specific CD4 (+) T cells obtained from an immunized donor.

The irradiated-sporozoite vaccine elicits sterile immunity againstPlasmodiumparasites in experimental rodent hosts and human volunteers. Based on rodent malaria models, it has been proposed that CD8+T cells are the key protective effector mechanism required in sporozoite-induced immunity. To investigate the role of class II-restricted immunity in protective immunity, we immunized β2-microglobulin knockout (β2M−/−) mice with irradiatedPlasmodium yoeliiorP. bergheisporozoites. Sterile immunity was obtained in the CD8+-T-cell-deficient mice immunized with eitherP. bergheiorP. yoeliisporozoites. β2M−/−mice with the BALB/c (H-2d) genetic background as well as those with the C57BL (H-2b) genetic background were protected. Effector mechanisms included CD4+T cells, mediated in part through the production of gamma interferon, and neutralizing antibodies that targeted the extracellular sporozoites. We conclude that in the absence of class I-restricted CD8+T cells, sporozoite-induced protective immunity can be effectively mediated by class II-restricted immune effector mechanisms. These results support efforts to develop subunit vaccines that effectively elicit high levels of antibody and CD4+T cells to targetPlasmodiumpreerythrocytic stages.

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. Trial Registration ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00587249","term_id":"NCT00587249"}}NCT00587249

Highly purified subunit vaccines require potent adjuvants in order to elicit optimal immune responses. In a previous phase I trial, an alum formulation of ICC-1132, a malaria vaccine candidate comprising hepatitis B core (HBc) virus-like particle containingPlasmodium falciparumcircumsporozoite (CS) protein epitopes, was shown to elicitPlasmodium falciparum-specific antibody and cellular responses. The present study was designed as a single-blind, escalating-dose phase I trial to evaluate the safety and immunogenicity of single intramuscular doses of ICC-1132 formulated in the more potent water-in-oil adjuvant Montanide ISA 720 (ICC-1132/ISA 720). The vaccine was safe and well tolerated, with transient injection site pain as the most frequent complaint. All vaccinees that received either 20 μg or 50 μg of ICC-1132/ISA 720 developed antiimmunogen and anti-HBc antibodies. The majority of volunteers in these two groups developed sporozoite-specific antibodies, predominantly of opsonizing immunoglobulin G subtypes. Peak titers and persistence of parasite-specific antibody following a single injection of the ISA 720 formulated vaccine were comparable to those obtained following two to three immunizations with alum-adsorbed ICC-1132. Peripheral blood mononuclear cells of ICC-1132/ISA 720 vaccinees proliferated and released cytokines (interleukin 2 and gamma interferon) when stimulated with recombinantP. falciparumCS protein, and CS-specific CD4+T-cell lines were established from volunteers with high levels of antibodies to the repeat region. The promising results obtained with a single dose of ICC-1132 formulated in Montanide ISA 720 encourage further clinical development of this malaria vaccine candidate.

ICC-1132 is a malaria vaccine candidate based on a modified hepatitis B virus core particle (HBc) bearing putative protective epitopes from the circumsporozoite protein (CS) of Plasmodium falciparum. While the epitope carrier itself is immunogenic, its potency can be increased by formulation with adjuvants. As a prelude to Phase I clinical trials, rhesus macaques were immunised twice with GMP grade ICC--1132 in saline or formulated with the adjuvants Alhydrogel (Alhydrogel) or Montanide((R)) ISA 720 (Montanide). Both adjuvant formulations gave significant humoral responses after the first injection, with titres increasing further after the second dose. The Montanide formulation was the most immunogenic, but undesirable reactogenicity in the form of sterile abscesses was associated with higher dosage levels of ICC--1132. These side effects could be avoided with lower antigen load, or by formulation of the second dose in Alhydrogel. Such measures also reduced peak titres and longevity of antibodies against CS, demonstrating the delicate balance between immunogenicity and reactogenicity of new vaccine formulations.

Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 ofPlasmodium vivax(PvMSP-1) and bind promiscuously to four different HLA-DRB1∗ alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previousP. vivaxinfections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with thePlasmodium falciparumimmunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restrictedP. falciparumT-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognizeP. vivaxmerozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion ofP. falciparumsporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.

A multiple antigen peptide (MAP) malaria vaccine containing minimal Plasmodium falciparum circumsporozoite protein repeat epitopes was assessed for safety and immunogenicity in volunteers of known class II genotypes. The MAP/alum/QS-21 vaccine formulation elicited high levels of parasite-specific antibodies in 10 of 12 volunteers expressing DQB1*0603, DRB1*0401, or DRB1*1101 class II molecules. In contrast, volunteers of other HLA genotypes were low responders or nonresponders. A second study of 7 volunteers confirmed the correlation of class II genotype and high responder phenotype. This is the first demonstration in humans that a peptide vaccine containing minimal T and B cell epitopes composed of only 5 amino acids (N, A, V, D, and P) can elicit antibody titers comparable to multiple exposures to irradiated P. falciparum-infected mosquitoes. Moreover, the high-responder phenotypes were predicted by analysis of peptide/HLA interactions in vitro, thus facilitating the rational design of epitope-based peptide vaccines for malaria, as well as for other pathogens.

Multiple antigen peptides (MAPs) containing epitopes of the major surface protein of the malaria sporozoite, the circumsporozoite (CS) protein, have been shown in previous studies to elicit antibody-mediated protection against sporozoite challenge in experimental murine and simian hosts. For the preparation for a phase I trial of a P. falciparum (T1B)4 MAP, which contains T and B cell epitopes from the CS repeat region, pre-clinical immunogenicity and adjuvant formulation studies were carried out in mice and Aotus monkeys. The (T1B)4 MAP was found to be immunogenic in three different species of owl monkeys, Aotus nancymae, A. vociferans and A. nigriceps. Optimal antibody responses were obtained in A. nancymae immunized s.c. with (T1B)4 MAP emulsified in Freund's, in which peak titers of over 10(6) were obtained in individual monkeys. MAP immunized A. vociferans also developed high levels of anti-sporozoite antibodies, although the kinetics and the magnitude of the response differed from A. nancymae. (T1B)4 MAP adsorbed to alum (aluminum hydroxide), a formulation that is acceptable for human use, was less immunogenic in naive A. nancymae, as well as A. nigriceps. The injection of MAPs/alum, however, significantly enhanced antibody responses in sporozoite-primed monkeys, suggesting that the administration of the MAP vaccine may be an effective means to increase the low levels of antibody present in individuals living in malaria endemic areas. The addition of a co-adjuvant QS-21, a purified saponin, significantly increased the immunogenicity of the alum-adsorbed MAP in both mice and monkeys, providing a vaccine formulation suitable for phase I trials in human volunteers.