Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

The CD4 T cell response to the tumor antigen Midkine was unknown. Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.

BackgroundEarly in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE).MethodsWe explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites.ResultsFrom 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8+ and 284 CD4+ T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides.ConclusionsOverall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes.

Paving the way for T cell-based immunotherapies in chronic hepatitis E

A viral T cell epitope was genetically inserted within the periplasmic MalE protein of Escherichia coli in two different permissive insertion sites and resulting hybrid proteins were used to study the in vitro and in vivo immunogenicity of the foreign T cell epitope. Purified hybrid MalE proteins containing the T cell epitope 120-132 (PreS:T) from PreS2 region of hepatitis B virus HBsAg inserted alone or with its adjacent B cell epitope (132-145) were able to induce strong peptide-specific T cell responses in mice. In vitro stimulation of primed lymph node cells or specific T cell hybridomas by the hybrid proteins required processing of the inserted T cell epitope and was inhibited by antigen-presenting cells fixation. The inserted T cell epitope was presented in vitro, in association with appropriate major histocompatibility complex molecules, as efficiently as free synthetic peptide. The in vitro immunogenicity of MalE hybrid proteins was increased by inserting four tandemly repeated copies of PreS:T, either at site 133 or 303. These results were confirmed in vivo by comparing the proliferative responses of lymph node cells from DBA/1 mice primed with MalE hybrid proteins containing one or four copies of PreS:T. Thus, the use of MalE hybrid proteins expressing multiple copies of a given foreign T cell epitope allows the induction of peptide-specific T cell response with a lower dose of priming antigen.

BackgroundLymphatic filariasis (LF), often referred to as elephantiasis, has been identified as one of the 17 neglected tropical diseases by the World Health Organization. Currently, there are no vaccines available to treat this infection in humans. Therefore, with the objective of devising a novel preventive measure, we exploited an immunoinformatics approach to design a multi-epitope-based subunit vaccine for LF, that can elicit a variety of immune responses within the host.In this study, different B cell, TC cell, and TH cell-binding epitopes were screened from the antigenic proteins of Brugia malayi and they were passed through several immunological filters to determine the optimal epitopes.ResultsAs a result, 15 CD8+, 3 CD4+, and 3 B cell epitopes were found to be prominent, antigenic, non-toxic, immunogenic and non-allergenic. The presence of conformational B cell epitopes and cytokine-inducing epitopes confirmed the humoral and cell-mediated immune response that would be triggered by the constructed vaccine model.Following that, the selected epitopes and TLR-4-specific adjuvant were ligated by appropriate peptide linkers to finalize the vaccine construct. Protein–protein docking of the vaccine structure with the TLR4 receptor predicted strong binding affinity and hence putatively confirms its ability to elicit an immune response. Further, the efficiency of the vaccine candidate to provide a long-lasting protective immunity was assessed by in silico immune simulation. The reverse translated vaccine sequence was also virtually cloned in the pET28a (+) plasmid after the optimization of the gene sequence.ConclusionSo taken together, by monitoring the overall in silico assessment, we hypothesize that our engineered peptide vaccine could be a viable prophylactic approach in the development of vaccines against the threat of human lymphatic filariasis.Graphical

A multi-epitope subunit vaccine based on CU/ZN-SOD, OMP31 and BP26 against Brucella melitensis infection in BALB/C mice

Bioinformatics-Guided structural characterization and immunogenicity assessment of multi-epitope vaccine candidates against Zika virus

T-cell based vaccines against SIV/HIV may reduce both transmission and disease progression by inducing broad and functionally relevant T cell responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency and virus replication. We have previously shown that a DNA vaccine (HIVBr18), encoding 18 HIV CD4 epitopes capable of binding to multiple HLA class II molecules was able to elicit broad, polyfunctional, and long-lived CD4+ and CD8+ T cell responses in BALB/c and multiple HLA class II transgenic mice. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that could be recognized across diverse common HLA class II alleles, this vaccine concept may cope with HIV-1 genetic variability and increase population coverage. Given the low immunogenicity of DNA vaccines in clinical trials, we tested the ability of a recombinant adenovirus serotype 5 encoding the 18 HIV epitopes (Ad5-HIVBr18) to increase specific cellular immune responses. We assessed the breadth and magnitude of HIV-specific proliferative and cytokine responses of CD4+ and CD8+ T cells induced by Ad5-HIVBr18 using different vaccination regimens/routes and compared to DNA immunization. Immunization with Ad5-HIVBr18 induced significantly higher specific CD4+ and CD8+ T cell proliferation, IFN-γ and TNF-α production than HIVBr18. The subcutaneous route of Ad5-HIVBr18 administration was associated with the highest responses. Ad5-HIVBr18 induced higher proliferative and cytokine responses than HIVBr18 up to 28 weeks post-immunization. Our results indicate that a vaccine based on an adenovirus vector encoding the HIVBr18 epitopes shows superior immunogenicity as compared to its DNA counterpart. These results support the possible testing of a vaccine encoding HIVBr18 in non-human primates and future clinical trials.

Effective immunotherapy for cancer depends on both cellular and humoral immune responses to tumor antigens. MUC1, which is expressed at increased levels on breast cancer, also exhibits altered glycosylation that contributes to the formation of novel antigens. The identification of MHC class I and II binding peptides derived from tumor-associated MUC1 has facilitated the development of MUC1 based cancer vaccines. We have shown that the MHC class I binding epitopes from MUC1 tandem repeat, when given as emulsification with adjuvants, has always resulted in strong cellular response with no antibody response. It is possible that better immunogenicity would be obtained using glycopeptides more representative of the novel forms of MUC1 as seen in cancer to which individuals may be less tolerant and that direct linking of the vaccine components would result in a superior immune response than delivering them as a mix. Here we show that a three-component vaccine composed of a TLR2 agonist, a helper epitope and a T cell epitope, which is also a B cell epitope, derived from the MUC1 can break tolerance and elicit both humoral and cellular immune responses. Immunization with the MUC1 glycopeptide vaccine led to a significant reduction in tumor burden compared to mice treated with adjuvants only and empty liposomes. The tri-component vaccine activated MUC1 glycopeptide-specific cytotoxic CD8+ T cells and elicited robust titers of IgG antibodies that lyse relevant tumor cells by ADCC.

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4+ T cells are important for the generation and maintenance of functional CD8+ cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4+ T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4+/CD8+ T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4+ and CD8+ T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4+ T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4+ T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4+ T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8+ T cells and antibody responses- elicited by other HIV immunogens.

While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-naïve and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-naïve individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-naïve vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-naïve counterparts.

BackgroundHuman Cytomegalovirus (HCMV) is a ubiquitous herpesvirus affecting approximately 90% of the world population. HCMV causes disease in immunologically naive and immunosuppressed patients. The prevention, diagnosis and therapy of HCMV infection are thus crucial to public health. The availability of effective prophylactic and therapeutic treatments remain a significant challenge and no vaccine is currently available. Here, we sought to define an epitope-based vaccine against HCMV, eliciting B and T cell responses, from experimentally defined HCMV-specific epitopes.ResultsWe selected 398 and 790 experimentally validated HCMV-specific B and T cell epitopes, respectively, from available epitope resources and apply a knowledge-based approach in combination with immunoinformatic predictions to ensemble a universal vaccine against HCMV. The T cell component consists of 6 CD8 and 6 CD4 T cell epitopes that are conserved among HCMV strains. All CD8 T cell epitopes were reported to induce cytotoxic activity, are derived from early expressed genes and are predicted to provide population protection coverage over 97%. The CD4 T cell epitopes are derived from HCMV structural proteins and provide a population protection coverage over 92%. The B cell component consists of just 3 B cell epitopes from the ectodomain of glycoproteins L and H that are highly flexible and exposed to the solvent.ConclusionsWe have defined a multiantigenic epitope vaccine ensemble against the HCMV that should elicit T and B cell responses in the entire population. Importantly, although we arrived to this epitope ensemble with the help of computational predictions, the actual epitopes are not predicted but are known to be immunogenic.

During the last decade, cancer vaccination has received a great deal of attention; however, documentation of the clinical effect remains limited. The introduction of immune checkpoint inhibitors as a novel therapeutic modality 1-4 could by combination increase the ability of a peptide vaccination to induce clinical responses. Survivin is an anti-apoptotic protein, first discovered in 1997 5. It is overexpressed in a wide array of cancers, including malignant melanoma (MM) 5, 6, and expression is correlated with disease progression and a poor prognosis in, for example colon cancer and neuroblastoma 7, 8. Survivin is an excellent target for cancer vaccines due to its universal expression in cancer; in addition, downregulation will impair the tumour cell's ability to proliferate and expand. Many T cell epitopes from human cancer antigens have relatively low binding affinities to class I human leucocyte antigens (HLAs) 9. To increase the immunogenicity of such peptides, amino acid substitutions have been introduced at anchor positions, but not at T cell receptor contact residues, to increase the peptide binding affinity to class I HLAs. Many of these modified peptides are more immunogenic than the native epitopes and have an enhanced capacity to induce specific T cell responses. Consequently, modified minimal epitopes have been used in many clinical vaccination studies. Several natural and modified epitopes of survivin have been extensively investigated for their immunogenicity 10-12, especially a modified analogue of peptide survivin (96–104), named Sur1M2 (LMLGEFLKL), with a methionine at position 2 instead of threonine. Sur1M2 binds with high affinity to the major histocompatibility complex (MHC) class I molecule, HLA-A2 13. The ability of Sur1M2 to induce specific CD8+ T cell responses has been confirmed both in vitro and in clinical settings 13, 14. The aim of vaccination is to induce a robust T cell response, especially of cytotoxic T lymphocytes, which are able to target and eradicate tumour cells. Most clinical trials have used short epitopes consisting of 8–11 amino acids to induce CD8+ T cell responses mediated by MHC class I molecules; however, CD4+ T cells can also be induced with short epitopes, for example MHC class I epitopes from Mycobacterium tuberculosis and influenza A peptides 15, 16. Recently, in a phase I trial, Groos et al. 17 vaccinated 53 patients with a cocktail of five survivin-derived minimal epitopes (9–10 amino acids) in Montanide ISA 51VG. They found that two peptides that were modified to increase the binding to HLA-A2 and HLA-A3 13, 18 were also recognized by HLA-DP4 and HLA-DR-restricted CD4 T cells. Thus, in many patients, Sur18K10 (RISTFKNWPK) and especially the Sur1M2 epitope described above were able to activate a specific CD4 T cell repertoire, in addition to activating CD8 T cell responses. We recently performed a similar trial at our centre, where patients with confirmed MM were injected with 250 μg of Sur1M2 and 250 μg of another T cell epitope from Indoleamine 2,3-dioxygenase 19 in a Montanide ISA-51 solution (www.clinicaltrials.gov; NCT01543464). Prior to vaccination, 75 μg of granulocyte macrophage colony-stimulating factor was injected and topical Imiquimod cream was applied the day before. The patients alternated between vaccination and temozolomide treatment every second week. Based on the data from Groos et al., we examined whether a similar activation of survivin-specific T cells could be detected in our patients. Hence, we selected a HLA-A2-positive patient with a strong Sur1M2 response, measured by enzyme-linked immunospot assay (ELISPOT) after vaccination (Fig. 1A). We stimulated peripheral blood mononuclear cells (PBMCs) each week with a log10 decreasing concentration of the peptide for 3 weeks. Hereafter, we sorted the cells using a tumour necrosis factor alpha (TNF-α) secretion assay (Miltenyi Biotec). The sorted cells were then expanded in a high-dose IL-2 rapid expansion protocol to increase the number of Sur1M2 cells. As depicted in Fig. 1B, intracellular cytokine staining (ICS) with Sur1M2 stimulation revealed that 17.4% of the CD4 T cells reacted towards Sur1M2. Importantly, Sur1M2-specific T cells cross-reacted with the wild-type Sur1 peptide (Sur1 WT, LTLGEFLKL). Next, we demonstrated that the CD4+ T cell response towards Sur1M2 was HLA class II mediated, as the addition of a pan HLA class II blocking antibody (Tü39) effectively decreased the frequency of CD4+ T cells producing TNF-α from 19.2% to 0.77% (Fig. 1C). Most importantly, we showed that the Sur1M2-specific CD4+ T cells recognized cells that intracellularly expressed survivin. Hence, we stimulated the Sur1M2-specific cells with autologous PBMCs electroporated with or without mRNA encoding survivin in an ICS assay. As depicted in Fig. 1D, the reaction of CD4 T cells was significantly stronger against PBMC transfected with survivin mRNA compared with mock transfection. To summarize, we confirm the data from Groos et al. and describe that the modified survivin epitope Sur1M2, used in many preclinical and clinical studies, not only is restricted to HLA-A2, but in addition is able to induce survivin-specific CD4+ T cells. Most importantly, we show that such T cells are functionally relevant as they recognize cells intracellularly expressing survivin. These findings imply that short peptides can boost CD4+ T cells. In addition, modified peptides with amino acid substitutions at anchor residues for specific HLA molecules, for example HLA-A2, may induce immune responses restricted to additional HLA molecules, even class II HLA molecules. Thus, it is important to consider potential HLA class II-restricted responses in future studies utilizing Sur1M2, as well as other modified T cell epitopes. This study was supported by the Danish Cancer Society, the Danish Council for Independent Research, Aase og Ejnar Danielsens Fond, Toyota Foundation and Herlev Hospital. The funders did not have a role in the writing of the article or the decision to submit the article for publication.

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.

The objective of this study was to elucidate the natural rodent host relationships of Guanarito and Pirital viruses (family Arenaviridae) in the plains of central Venezuela. Ninety-two arenavirus isolates from 607 animals, representing 10 different rodent species, were characterized to the level of serotype. The 92 isolates comprised 19 Guanarito virus strains and 73 Pirital virus strains. The 19 Guanarito virus isolates were from Zygodontomys brevicauda; 72 (98.6%) of the 73 Pirital virus isolates were from Sigmodon alstoni. These results indicate that the natural rodent associations of these 2 sympatric arenaviruses are highly specific and that Z brevicauda and S. alstoni are the principal rodent hosts of Guanarito and Pirital viruses, respectively.