Rotavirus specific plasma secretory immunoglobulin in children with acute gastroenteritis and children vaccinated with an attenuated human rotavirus vaccine.

Rotavirus (RV)-specific secretory immunoglobulin (RV-SIg) has been previously detected in serum of naturally RV infected children and shown to reflect the intestinal Ig immune response. Total plasma SIgA and plasma RV-SIg were evaluated by ELISA in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-SIg was only detected in children with evidence of previous RV infection or with acute RV gastroenteritis. Vaccinees had higher RV-SIg titers than placebo recipients and RV-SIg titers increased after the second vaccine dose. RV-SIg measured after the second dose correlated with protection when vaccinees and placebo recipients were analyzed jointly. RV-SIg may serve as a valuable correlate of protection for RV vaccines.

Do antibody responses to malaria vaccine candidates influenced by the level of malaria transmission protect from malaria?

OBJECTIVES: To examine whether the humoural response to malaria vaccine candidate antigens, Plasmodium falciparum [circumsporozoite repetitive sequence (NANP)(5) GLURP fragments (R0 and R2) and MSP3] varies with the level of malaria transmission and to determine whether the antibodies (IgG) present at the beginning of the malaria transmission season protect against clinical malaria. METHODS: Cross-sectional surveys were conducted to measure antibody response before, at the peak and at the end of the transmission season in children aged 6 months to 10 years in two villages with different levels of malaria transmission. A cohort study was performed to estimate the incidence of clinical malaria. RESULTS: Antibodies to these antigens showed different seasonal patterns. IgG concentrations to any of the four antigens were higher in the village with high entomological inoculation rate. Multivariate analysis of combined data from the two villages indicated that children who were classified as responders to the selected antigens were at lower risk of clinical malaria than children classified as non-responders [(NANP)(5) (incidence rate ratio (IRR) = 0.65, 95% CI: 0.46-0.92; P = 0.016), R0 (IRR = 0.69, 95% CI: 0.48-0.97; P = 0.032), R2 (IRR = 0.73, 95% CI: 0.50-1.06; P = 0.09), MSP3 (IRR = 0.52, 95% CI: 0.32-0.85; P = 0.009)]. Fitting a model with all four antibody responses showed that MSP3 looked the best malaria vaccine candidate (IRR = 0.63; 95% CI: 0.38-1.05; P = 0.08). CONCLUSION: Antibody levels to the four antigens are affected by the intensity of malaria transmission and associated with protection against clinical malaria. It is worthwhile investing in the development of these antigens as potential malaria vaccine candidates.

Technology transfer of an oil-in-water vaccine-adjuvant for strengthening pandemic influenza preparedness in Indonesia.

With the current enzootic circulation of highly pathogenic avian influenza viruses, the ability to increase global pandemic influenza vaccine production capacity is of paramount importance. This has been highlighted by, and is one of the main pillars of, the WHO Global Action Plan for Influenza Vaccines (GAP). Such capacity expansion is especially relevant in developing countries. The Vaccine Formulation Laboratory at University of Lausanne is engaged in the technology transfer of an antigen-sparing oil-in-water adjuvant in order to empower developing countries vaccine manufacturers to increase pandemic influenza vaccine capacity. In a one-year project funded by United States Department of Health and Human Services, the Vaccine Formulation Laboratory transferred the process know-how and associated equipment for the pilot-scale manufacturing of an oil-in-water adjuvant to Bio Farma, Indonesia's state-owned vaccine manufacturer, for subsequent formulation with H5N1 pandemic influenza vaccines. This paper describes the experience acquired and lessons learnt from this technology transfer project.

Innate immune response to poxvirus vectors

Summary : A large body of evidence indicates that the innate immune system plays a key role in host response to viral infection. Recently, Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptor receptors (NLRs) have emerged as key innate immune sensors of microbial products, eliciting intracellular signaling and leading to the production of chemokines, cytokines and interferons (IFNs) that shape innate immune responses and coordinate the development of adaptive immunity. Poxviruses are currently developed as vaccines vectors for infectious diseases such as HIV, tuberculosis and malaria. Modified vaccinia virus Ankara (MVA) and New York vaccinia virus (NWAC) are attenuated, replication deficient strains of poxvirus. The mechanisms underlying innate immune responses to MVA and NYVAC are poorly characterized. Thus, the objectives of the project were to determine the innate immune profile stimulated by poxviruses in innate immune cells and to evaluate the impact of modifications in the viral genome on MVA and NYVAC immunogenicity. MVA stimulated the production of abundant amounts of chemokines and IFNß but low levels of cytokines by human macrophages. In contrast, NYVAC weakly stimulated the production of all mediators. Interestingly, MVA and NYVAC strongly stimulated innate immune responses in vivo and in human whole blood, suggesting that a soluble factors}, possibly a complement component, was required for optimal activation of innate immune cells by poxviruses. Modified MVA and NYVAC produced by single or multiple deletions of viral genes targeting crucial pathways of host innate immunity, and mutant poxviruses with limited replication capacity, increased the production of pro-inflammatory molecules by human whole blood. Gene expression profiling in human macrophages confirmed the increased immunologic stimulatory capacity of modified poxviruses. The pathways activated by MVA and NYVAC in innate immune cells were described by analysing the response of knockdown or shRNA transduced macrophages with impaired expression of TLRs and their adaptors (MyD8$ and TRIF), RLRs (RIG-I, MDA-5 and the adaptor IPS-1) and the NALP3 inflammasome composed óf the NLR NALP3, caspase-1 and ASC. These experiments revealed a critical role for TLR2-TLR6-MyD88 in the production of tFNß-independent chemokines and of MDA-5-IPS-1 in the production of IFNß and IFNßdependent chemokines. The transcription of the iL1b gene encoding for the IL-1ß cytokine was initiated through TLR2-MyD88, whereas the maturation and the secretion of IL-1ß were controlled by the NALP3 inflammasome. Finally, we analyzed the role of macrophage migration inhibitory factor (MIF), a mediator of inflammation and innate immune responses, in MVA infection. We observed that MVA infection increased MIF production by innate immune cells and that MIF deficiency impaired macrophage and dendritic cell responses (ie migration, maturation, cytokine and IFN production) to MVA infection in vitro and in vivo. Moreover, MIF-deficiency resulted in delayed anti-MVA specific antibody production in mice immunized with the virus. In conclusion, we demonstrate. that poxviruses can be modified genetically to improve their immunogenicity. We also report the first comprehensive analysis of poxvirus sensing by innate immune cells, showing that the TLR, RLR and NLR pathways play specific and coordinated roles in regulating cytokine, chemokine and IFN response to poxvirus infection. Finally, we show that MIF is an integral host component involved in innate and adaptive immune responses to MVA infection. The present findings provide important information relevant to the study of the pathogenesis of poxvirus infections and allow a better understanding of the immunogenic potential of vaccine vectors, which is required for the development of optimized modìfied pox-vaccine vectors.

Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4(+) T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

EV02: a Phase I trial to compare the safety and immunogenicity of HIV DNA-C prime-NYVAC-C boost to NYVAC-C alone.

The aim of this randomised controlled trial was to see if the addition of 4 mg/ml DNA-C priming given by the intramuscular route at weeks 0 and 4 to NYVAC-C at weeks 20 and 24, safely increased the proportion of participants with HIV-specific T-cell responses measured by the interferon (IFN)-gamma ELISpot assay at weeks 26 and/or 28 compared to NYVAC-C alone. Although 2 individuals discontinued after the first DNA-C due to adverse events (1 vaso-vagal; 1 transient, asymptomatic elevation in alanine transaminase), the vaccines were well tolerated. Three others failed to complete the regimen (1 changed her mind; 2 lost to follow-up). Of the 35 that completed the regimen 90% (18/20) in the DNA-C group had ELISpot responses compared to 33% (5/15) that received NYVAC-C alone (p=0.001). Responses were to envelope in the majority (21/23). Of the 9 individuals with responses to envelope and other peptides, 8 were in the DNA-C group. These promising results suggest that DNA-C was an effective priming agent, that merits further investigation.

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.

A novel Th cell epitope of Candida albicans mediates protection from fungal infection.

Fungal pathogens are a frequent cause of opportunistic infections. They live as commensals in healthy individuals but can cause disease when the immune status of the host is altered. T lymphocytes play a critical role in pathogen control. However, specific Ags determining the activation and function of antifungal T cells remain largely unknown. By using an immunoproteomic approach, we have identified for the first time, to our knowledge, a natural T cell epitope from Candida albicans. Isolation and sequencing of MHC class II-bound ligands from infected dendritic cells revealed a peptide that was recognized by a major population of all Candida-specific Th cells isolated from infected mice. Importantly, human Th cells also responded to stimulation with the peptide in an HLA-dependent manner but without restriction to any particular HLA class II allele. Immunization of mice with the peptide resulted in a population of epitope-specific Th cells that reacted not only with C. albicans but also with other clinically highly relevant species of Candida including the distantly related Candida glabrata. The extent of the reaction to different Candida species correlated with their degree of phylogenetic relationship to C. albicans. Finally, we show that the newly identified peptide acts as an efficient vaccine when used in combination with an adjuvant inducing IL-17A secretion from peptide-specific T cells. Immunized mice were protected from fatal candidiasis. Together, these results uncover a new immune determinant of the host response against Candida ssp. that could be exploited for the development of antifungal vaccines and immunotherapies.

Quoi de neuf en medecine des voyages? [What's new in travel medicine?]

Les menaces pour la santé des voyageurs proviennent souvent de l'émergence ou de la réémergence d'anciennes et de nouvelles maladies infectieuses. En Amérique du Sud c'est une augmentation des cas de fièvre jaune qui a fait du bruit. A Bornéo, une nouvelle espèce de Plasmodium pathogène pour l'humain a été mise en évidence. Après l'épidémie sur les îles de l'océan Indien, le virus du Chikungunya a causé de nouveaux foyers et ceci même en Europe. La première journée mondiale de la rage a rappelé que cette maladie continue à tuer un nombre important de personnes dans les pays aux ressources limitées. La rage affecte également des personnes de nos pays comme l'illustre deux situations récentes. Finalement, le nouveau Règlement sanitaire international permettra à l'OMS de mieux répondre aux urgences de santé publique de portée internationale. The threats for the health of travellers come often from the emergence or the re-emergence of old and new infectious diseases. In South America an important increase of the number of cases of yellow fever was reported. On the island of Borneo a new species of Plasmodium pathogenic for humans has been identified. After the Chikungunya epidemic that affected the islands of the Indian Ocean, the virus has caused new epidemic foci and this even in Europe. The first World Rabies Day reminded us that this disease still kills a large number of persons in countries with limited resources. Rabies can also affect persons from our countries, as it was illustrated by two recent situations. Finally the new International Health Regulation will allow the WHO to better respond to public health emergencies of international concern

Virus-like particles induce robust human T-helper cell responses.

Among synthetic vaccines, virus-like particles (VLPs) are used for their ability to induce strong humoral responses. Very little is reported on VLP-based-vaccine-induced CD4(+) T-cell responses, despite the requirement of helper T cells for antibody isotype switching. Further knowledge on helper T cells is also needed for optimization of CD8(+) T-cell vaccination. Here, we analysed human CD4(+) T-cell responses to vaccination with MelQbG10, which is a Qβ-VLP covalently linked to a long peptide derived from the melanoma self-antigen Melan-A. In all analysed patients, we found strong antibody responses of mainly IgG1 and IgG3 isotypes, and concomitant Th1-biased CD4(+) T-cell responses specific for Qβ. Although less strong, comparable B- and CD4(+) T-cell responses were also found specific for the Melan-A cargo peptide. Further optimization is required to shift the response more towards the cargo peptide. Nevertheless, the data demonstrate the high potential of VLPs for inducing humoral and cellular immune responses by mounting powerful CD4(+) T-cell help.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells.

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3₁₄₀₆₋₁₄₁₅ and NS5B₂₅₉₄₋₂₆₀₂). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

The Vaccine Formulation Laboratory: a platform for access to adjuvants.

Adjuvants are increasingly used by the vaccine research and development community, particularly for their ability to enhance immune responses and for their dose-sparing properties. However, they are not readily available to the majority of public sector vaccine research groups, and even those with access to suitable adjuvants may still fail in the development of their vaccines because of lack of knowledge on how to correctly formulate the adjuvants. This shortcoming led the World Health Organization to advocate for the establishment of the Vaccine Formulation Laboratory at the University of Lausanne, Switzerland. The primary mission of the laboratory is to transfer adjuvants and formulation technology free of intellectual property rights to academic institutions, small biotechnology companies and developing countries vaccine manufacturers. In this context, the transfer of an oil-in-water emulsion to Bio Farma, an Indonesian vaccine manufacturer, was initiated to increase domestic pandemic influenza vaccine production capacity as part of the national pandemic influenza preparedness plan.

Neoadjuvant and adjuvant strategies in renal cell carcinoma: more questions than answers.

The current standard treatment for early stage (I-III) renal cell cancer (RCC) is surgery. While the prognosis of stage I tumors is excellent, stage II and particularly stage III have a high risk of relapse. The adjuvant treatment of patients with RCC remains an area of investigation, with patient selection being a key aspect. There are currently two prognostic nomograms to establish the risk of relapse in patients with resected RCC. The results of earlier studies of adjuvant therapy, including the use chemotherapy and/or immunotherapy after nephrectomy have failed to show any benefit in the outcome of patients at risk of developing local recurrence or distant metastases. Two recent phase III trials with vaccines (autologous tumor cell vaccine and autologous tumor-derived heat shock protein peptide complex-96) have shown promising, albeit still preliminary, results. In the metastatic RCC setting, recent advances in the molecular understanding of oncogenic pathways have led to the development of new therapeutic strategies with the use of targeted therapies in the adjuvant setting. Neoadjuvant treatment is another treatment modality currently being evaluated for patients with early disease and in patients with metastatic RCC with inoperable primary tumors. The questions that remain unanswered include activity of these agents in early stages of the disease, patient selection, optimal start time of the adjuvant treatment, and finally, the optimal length of treatment.