Leishmania cysteine proteinases: from gene to subunit vaccine.

Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a Cterminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB / c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.

A Candidate HIV/AIDS Vaccine (MVA-B) Lacking Vaccinia Virus Gene C6L Enhances Memory HIV-1-Specific T-Cell Responses.

The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8(+) T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8(+) T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8(+) T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.

Deletion of the viral anti-apoptotic gene F1L in the HIV/AIDS vaccine candidate MVA-C enhances immune responses against HIV-1 antigens.

Vaccinia virus (VACV) encodes an anti-apoptotic Bcl-2-like protein F1 that acts as an inhibitor of caspase-9 and of the Bak/Bax checkpoint but the role of this gene in immune responses is not known. Because dendritic cells that have phagocytosed apoptotic infected cells cross-present viral antigens to cytotoxic T cells inducing an antigen-specific immunity, we hypothesized that deletion of the viral anti-apoptotic F1L gene might have a profound effect on the capacity of poxvirus vectors to activate specific immune responses to virus-expressed recombinant antigens. This has been tested in a mouse model with an F1L deletion mutant of the HIV/AIDS vaccine candidate MVA-C that expresses Env and Gag-Pol-Nef antigens (MVA-C-ΔF1L). The viral gene F1L is not required for virus replication in cultured cells and its deletion in MVA-C induces extensive apoptosis and expression of immunomodulatory genes in infected cells. Analysis of the immune responses induced in BALB/c mice after DNA prime/MVA boost revealed that, in comparison with parental MVA-C, the mutant MVA-C-ΔF1L improves the magnitude of the HIV-1-specific CD8 T cell adaptive immune responses and impacts on the CD8 T cell memory phase by enhancing the magnitude of the response, reducing the contraction phase and changing the memory differentiation pattern. These findings reveal the immunomodulatory role of F1L and that the loss of this gene is a valid strategy for the optimization of MVA as vaccine vector.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant.

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.

Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

The serine repeat antigen (SERA) gene family phylogeny in Plasmodium: the impact of GC content and reconciliation of gene and species trees.

Plasmodium falciparum is the parasite responsible for the most acute form of malaria in humans. Recently, the serine repeat antigen (SERA) in P. falciparum has attracted attention as a potential vaccine and drug target, and it has been shown to be a member of a large gene family. To clarify the relationships among the numerous P. falciparum SERAs and to identify orthologs to SERA5 and SERA6 in Plasmodium species affecting rodents, gene trees were inferred from nucleotide and amino acid sequence data for 33 putative SERA homologs in seven different species. (A distance method for nucleotide sequences that is specifically designed to accommodate differing GC content yielded results that were largely compatible with the amino acid tree. Standard-distance and maximum-likelihood methods for nucleotide sequences, on the other hand, yielded gene trees that differed in important respects.) To infer the pattern of duplication, speciation, and gene loss events in the SERA gene family history, the resulting gene trees were then "reconciled" with two competing Plasmodium species tree topologies that have been identified by previous phylogenetic studies. Parsimony of reconciliation was used as a criterion for selecting a gene tree/species tree pair and provided (1) support for one of the two species trees and for the core topology of the amino acid-derived gene tree, (2) a basis for critiquing fine detail in a poorly resolved region of the gene tree, (3) a set of predicted "missing genes" in some species, (4) clarification of the relationship among the P. falciparum SERA, and (5) some information about SERA5 and SERA6 orthologs in the rodent malaria parasites. Parsimony of reconciliation and a second criterion--implied mutational pattern at two key active sites in the SERA proteins-were also seen to be useful supplements to standard "bootstrap" analysis for inferred topologies.

Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1.

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses

The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon gamma enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/10(6) mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen

Impact of Pre-Existing Immunity on the Selection of Rare Adenovirus Vector Candidates: Implications for HIV Vaccine Development

Background: Adenovirus serotype 5 (Ad5) phase IIb vaccine trial (STEP) was prematurely stopped due to a lack of efficacy and two-fold higher incidence of HIV infection among Ad5 seropositive vaccine recipients. We have recently demonstrated that Ad5 immune complexes (Ad5 ICs)-mediated activation of the dendritic cell (DC)-T cell axis was associated with the enhancement of HIV infection in vitro. Although the direct role of Ad5 neutralizing antibodies (NAbs) in the increase of HIV susceptibility during the STEP trial is still under debate, vector-specific NAbs remain a major hurdle for vector-based gene therapies or vaccine strategies. To surmount this obstacle, vectors based on ''rare'' Ad serotypes including Ad6, Ad26, Ad36 and Ad41 were engineered.Methods: The present study aimed to determine whether Ad ICmediated DC maturation could be circumvented using these Advector candidates.Results: We found that all Ad vectors tested forming ICs with plasma containing serotype-specific NAbs had the capacity to 1) mature human DCs as monitored by the up-regulation of costimulatory molecules and the release of pro-inflammatory cytokines (TNF-a), via the stabilization of Ad capsid at endosomal but not lysosomal pH rendering Ad DNA/TLR9 interactions possible and 2) potentiate Ad-specific CD4 and CD8 T cell responses.Conclusion: In conclusion, despite a conserved DC maturation potential, the low prevalence of serotype-specific NAbs renders rare Ad vectors attractive for vaccine strategies.

Intravaginal TLR agonists increase local vaccine-specific CD8 T cells and human papillomavirus-associated genital-tumor regression in mice.

Human papillomaviruses (HPV)-related cervical cancer is the second leading cause of cancer death in women worldwide. Despite active development, HPV E6/E7 oncogene-specific therapeutic vaccines have had limited clinical efficacy to date. Here, we report that intravaginal (IVAG) instillation of CpG-ODN (TLR9 agonist) or poly-(I:C) (TLR3 agonist) after subcutaneous E7 vaccination increased ∼fivefold the number of vaccine-specific interferon-γ-secreting CD8 T cells in the genital mucosa (GM) of mice, without affecting the E7-specific systemic response. The IVAG treatment locally increased both E7-specific and total CD8 T cells, but not CD4 T cells. This previously unreported selective recruitment of CD8 T cells from the periphery by IVAG CpG-ODN or poly-(I:C) was mediated by TLR9 and TLR3/melanoma differentiation-associated gene 5 signaling pathways, respectively. For CpG, this recruitment was associated with a higher proportion of GM-localized CD8 T cells expressing both CCR5 and CXCR3 chemokine receptors and E-selectin ligands. Most interestingly, IVAG CpG-ODN following vaccination led to complete regression of large genital HPV tumors in 75% of mice, instead of 20% with vaccination alone. These findings suggest that mucosal application of immunostimulatory molecules might substantially increase the effectiveness of parenterally administered vaccines.Mucosal Immunology advance online publication 12 September 2012; doi:10.1038/mi.2012.83.

The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen.

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.

Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.

Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses.

Correlates of immune-mediated protection to most viral and cancer vaccines are still unknown. This impedes the development of novel vaccines to incurable diseases such as HIV and cancer. In this study, we have used functional genomics and polychromatic flow cytometry to define the signature of the immune response to the yellow fever (YF) vaccine 17D (YF17D) in a cohort of 40 volunteers followed for up to 1 yr after vaccination. We show that immunization with YF17D leads to an integrated immune response that includes several effector arms of innate immunity, including complement, the inflammasome, and interferons, as well as adaptive immunity as shown by an early T cell response followed by a brisk and variable B cell response. Development of these responses is preceded, as demonstrated in three independent vaccination trials and in a novel in vitro system of primary immune responses (modular immune in vitro construct [MIMIC] system), by the coordinated up-regulation of transcripts for specific transcription factors, including STAT1, IRF7, and ETS2, which are upstream of the different effector arms of the immune response. These results clearly show that the immune response to a strong vaccine is preceded by coordinated induction of master transcription factors that lead to the development of a broad, polyfunctional, and persistent immune response that integrates all effector cells of the immune system.