Immunization with Mycobacterium tuberculosis-Specific Antigens Bypasses T Cell Differentiation from Prior Bacillus Calmette-Guérin Vaccination and Improves Protection in Mice.

Despite the fact that the majority of people in tuberculosis (TB)-endemic areas are vaccinated with the Bacillus Calmette-Guérin (BCG) vaccine, TB remains the leading infectious cause of death. Data from both animal models and humans show that BCG and subunit vaccines induce T cells of different phenotypes, and little is known about how BCG priming influences subsequent booster vaccines. To test this, we designed a novel Mycobacterium tuberculosis-specific (or "non-BCG") subunit vaccine with protective efficacy in both mice and guinea pigs and compared it to a known BCG boosting vaccine. In naive mice, this M. tuberculosis-specific vaccine induced similar protection compared with the BCG boosting vaccine. However, in BCG-primed animals, only the M. tuberculosis-specific vaccine added significantly to the BCG-induced protection. This correlated with the priming of T cells with a lower degree of differentiation and improved lung-homing capacity. These results have implications for TB vaccine design.

HCV p7 as a novel vaccine-target inducing multifunctional CD4+ and CD8+ T-cells targeting liver cells expressing the viral antigen.

Despite recent treatment advances for chronic hepatitis C virus (HCV) infection, a vaccine is urgently needed for global control of this important liver pathogen. The lack of robust immunocompetent HCV infection models makes it challenging to identify correlates of protection and test vaccine efficacy. However, vigorous CD4+ and CD8+ T-cell responses are detected in patients that spontaneously resolve acute infection, whereas dysfunctional T-cell responses are a hallmark of chronic infection. The HCV p7 protein, forming ion-channels essential for viral assembly and release, has not previously been pursued as a vaccine antigen. Herein, we demonstrated that HCV p7 derived from genotype 1a and 1b sequences are highly immunogenic in mice when employed as overlapping peptides formulated as nanoparticles with the cross-priming adjuvant, CAF09. This approach induced multifunctional cytokine producing CD4+ and CD8+ T-cells targeting regions of p7 that are subject to immune pressure during HCV infection in chimpanzees and humans. Employing a surrogate in vivo challenge model of liver cells co-expressing HCV-p7 and GFP, we found that vaccinated mice cleared transgene expressing cells. This study affirms the potential of a T-cell inducing nanoparticle vaccine platform to target the liver and introduces HCV p7 as a potential target for HCV vaccine explorations.

Cyclooxygenase inhibitors impair CD4 T cell immunity and exacerbate Mycobacterium tuberculosis infection in aerosol-challenged mice.

Tuberculosis, caused by infection with Mycobacterium tuberculosis (Mtb), kills over 1.6 million people each year despite availability of antibiotics. The increase in drug resistant Mtb strains is a major public health emergency and host-directed therapy as adjunct to antibiotic treatment has gained increased interest. Cyclooxygenase inhibitors (COXi) are frequently used drugs to alleviate tuberculosis related symptoms. Mouse studies of acute intravenous Mtb infection have suggested a potential benefit of COXi for host-directed therapy. Here we show that COXi treatment (ibuprofen and celecoxib) is detrimental to Mtb control in different mouse models of respiratory infection. This effect links to impairments of the Type-1 helper (Th1) T-cell response as CD4 T-cells in COXi-treated animals have significantly decreased Th1 differentiation, reduced IFNγ expression and decreased protective capacity upon adoptive transfer. If confirmed in clinical trials, these findings could have major impact on global health and question the use of COXi for host-directed therapy.

Mucosal boosting of H56:CAF01 immunization promotes lung-localized T cells and an accelerated pulmonary response to Mycobacterium tuberculosis infection without enhancing vaccine protection.

T cell-mediated protection against Mycobacterium tuberculosis (Mtb) is dependent upon the ability to localize within the site of pulmonary infection and directly interact with infected cells. In turn, vaccine strategies to improve rapid T cell targeting of Mtb-infected cells after pulmonary exposure are being actively pursued. Given parenterally, the subunit vaccine H56:CAF01 elicits polyfunctional CD4 T cells that localize to the lung parenchyma and confer durable protection. Here, we find that airway mucosal boosting of parenteral H56:CAF01 immunization greatly enhances the population of long-lived lung-resident T cells (Trm) and increases early vaccine T cell responses to pulmonary Mtb challenge in multiple mouse models. However, mucosal boosting does not alter the Th1/17 vaccine signature typical of H56:CAF01 and does not further improve durable control of pulmonary infection following aerosol Mtb-challenge. Additional mucosal boosting with H56:CAF01 further enhances the Trm response without further improving protection, while blocking the recruitment of non-Trm with FTY720-treatment failed to exposed Trm-mediated protection in mucosally boosting animals. These results demonstrate the limitations of maximizing lung-localized Trm in vaccine control of pulmonary Mtb infection, especially within an immunization protocol that is already optimized for the induction of mucosal-homing Th17 cells.

T Cells Primed by Live Mycobacteria Versus a Tuberculosis Subunit Vaccine Exhibit Distinct Functional Properties.

Despite inducing strong T cell responses, Mycobacterium tuberculosis (Mtb) infection fails to elicit protective immune memory. As such latently infected or successfully treated Tuberculosis (TB) patients are not protected against recurrent disease. Here, using a mouse model of aerosol Mtb infection, we show that memory immunity to H56/CAF01 subunit vaccination conferred sustained protection in contrast to the transient natural immunity conferred by Mtb infection. Loss of protection to re-infection in natural Mtb memory was temporally linked to an accelerated differentiation of ESAT-6- and to a lesser extent, Ag85B-specific CD4 T cells in both the lung parenchyma and vasculature. This phenotype was characterized by high KLRG1 expression and low, dual production of IFN-γ and TNF. In contrast, H56/CAF01 vaccination elicited cells that expressed low levels of KLRG1 with copious expression of IL-2 and IL-17A. Co-adoptive transfer studies revealed that H56/CAF01 induced memory CD4 T cells efficiently homed into the lung parenchyma of mice chronically infected with Mtb. In comparison, natural Mtb infection- and BCG vaccine-induced memory CD4 T cells exhibited a poor ability to home into the lung parenchyma. These studies suggest that impaired lung migratory capacity is an inherent trait of the terminally differentiated memory responses primed by mycobacteria/mycobacterial vectors.

The dynamics of immune responses to Mycobacterium tuberculosis during different stages of natural infection: A longitudinal study among Greenlanders.

OBJECTIVE: Understanding human immunity to Mycobacterium tuberculosis (Mtb) during different stages of infection is important for development of an effective tuberculosis (TB) vaccine. We aimed to evaluate immunity to Mtb infection by measuring immune responses to selected Mtb antigens expressed during different stages of infection over time and to observe sustainability of immunity. METHODS: In a cohort study comprising East Greenlanders aged 17-22 years (2012 to 2014) who had either; undetectable Mtb infection, ongoing or prior Mtb infection at enrolment, we measured immunity to 15 antigens over a one-year period. Quantiferon-TB Gold testing (QFT) defined Mtb infection status (undetected/detected). The eligible study population of East Greenlanders aged 17-22 years was identified from the entire population using the Civil Registration System. From the source population 65 participants were selected by stratified random sampling according to information on Mtb infection stage. Retrospective and prospective information on notified TB (including treatment) was obtained through the mandatory TB notification system and was used to characterise Mtb infection stage (ongoing/prior). Immunity to 15 antigens including two QFT antigens, PPD and 12 non-QFT antigens (representing early, constitutive and latent Mtb infection) was assessed by measuring immune responses using whole-blood antigen stimulation and interferon gamma measurement. RESULTS: Of 65 participants, 54 were considered Mtb-infected. Immunity to Mtb infection fluctuated with high annual risk of conversion (range: 6-69%) and reversion (range: 5-95%). During follow-up, five (8%) participants were notified with TB; neither conversion nor reversion was associated with an increased risk of progressing to TB. CONCLUSIONS: Our findings suggest that human immunity to natural Mtb infection over time is versatile with fluctuations, resulting in high levels of conversion and reversion of immunity, thus human immunity to Mtb is much more dynamic than anticipated. The study findings suggest future use of longitudinal assessment of immune responses when searching for TB vaccine candidate antigens.

Broadening CD4+ and CD8+ T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes.

Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4+ and CD8+ T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4+ T cells, whereas the NS3 pepmix induced a far more vigorous CD4+ T cell response and was as potent a CD8+ T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ+) tumor necrosis factor alpha-positive (TNF-α+) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity.IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4+ and CD8+ T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4+ and CD8+ T cells compared to vaccination with rNS3, which generated only CD4+ T cell responses.

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection.

Low Antigen Dose in Adjuvant-Based Vaccination Selectively Induces CD4 T Cells with Enhanced Functional Avidity and Protective Efficacy.

T cells with high functional avidity can sense and respond to low levels of cognate Ag, a characteristic that is associated with more potent responses against tumors and many infections, including HIV. Although an important determinant of T cell efficacy, it has proven difficult to selectively induce T cells of high functional avidity through vaccination. Attempts to induce high-avidity T cells by low-dose in vivo vaccination failed because this strategy simply gave no response. Instead, selective induction of high-avidity T cells has required in vitro culturing of specific T cells with low Ag concentrations. In this study, we combined low vaccine Ag doses with a novel potent cationic liposomal adjuvant, cationic adjuvant formulation 09, consisting of dimethyldioctadecylammonium liposomes incorporating two immunomodulators (monomycolyl glycerol analog and polyinosinic-polycytidylic acid) that efficiently induces CD4 Th cells, as well as cross-primes CD8 CTL responses. We show that vaccination with low Ag dose selectively primes CD4 T cells of higher functional avidity, whereas CD8 T cell functional avidity was unrelated to vaccine dose in mice. Importantly, CD4 T cells of higher functional avidity induced by low-dose vaccinations showed higher cytokine release per cell and lower inhibitory receptor expression (PD-1, CTLA-4, and the apoptosis-inducing Fas death receptor) compared with their lower-avidity CD4 counterparts. Notably, increased functional CD4 T cell avidity improved antiviral efficacy of CD8 T cells. These data suggest that potent adjuvants, such as cationic adjuvant formulation 09, render low-dose vaccination a feasible and promising approach for generating high-avidity T cells through vaccination.

High Antigen Dose Is Detrimental to Post-Exposure Vaccine Protection against Tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), causes 1.8M deaths annually. The current vaccine, BCG, has failed to eradicate TB leaving 25% of the world's population with latent Mtb infection (LTBI), and 5-10% of these people will reactivate and develop active TB. An efficient therapeutic vaccine targeting LTBI could have an enormous impact on global TB incidence, and could be an important aid in fighting multidrug resistance, which is increasing globally. Here we show in a mouse model using the H56 (Ag85B-ESAT-6-Rv2660) TB vaccine candidate that post-exposure, but not preventive, vaccine protection requires low vaccine antigen doses for optimal protection. Loss of protection from high dose post-exposure vaccination was not associated with a loss of overall vaccine response magnitude, but rather with greater differentiation and lower functional avidity of vaccine-specific CD4 T cells. High vaccine antigen dose also led to a decreased ability of vaccine-specific CD4 T cells to home into the Mtb-infected lung parenchyma, a recently discovered important feature of T cell protection in mice. These results underscore the importance of T cell quality rather than magnitude in TB-vaccine protection, and the significant role that antigen dosing plays in vaccine-mediated protection.

Comparative Systems Analyses Reveal Molecular Signatures of Clinically tested Vaccine Adjuvants.

A better understanding of the mechanisms of action of human adjuvants could inform a rational development of next generation vaccines for human use. Here, we exploited a genome wide transcriptomics analysis combined with a systems biology approach to determine the molecular signatures induced by four clinically tested vaccine adjuvants, namely CAF01, IC31, GLA-SE and Alum in mice. We report signature molecules, pathways, gene modules and networks, which are shared by or otherwise exclusive to these clinical-grade adjuvants in whole blood and draining lymph nodes of mice. Intriguingly, co-expression analysis revealed blood gene modules highly enriched for molecules with documented roles in T follicular helper (TFH) and germinal center (GC) responses. We could show that all adjuvants enhanced, although with different magnitude and kinetics, TFH and GC B cell responses in draining lymph nodes. These results represent, to our knowledge, the first comparative systems analysis of clinically tested vaccine adjuvants that may provide new insights into the mechanisms of action of human adjuvants.

Host immunity to Mycobacterium tuberculosis and risk of tuberculosis: A longitudinal study among Greenlanders.

BACKGROUND: Human immune responses to latent Mycobacterium tuberculosis (Mtb) infection (LTBI) may enable individuals to control Mtb infection and halt progression to tuberculosis (TB), a hypothesis applied in several novel TB vaccines. We aimed to evaluate whether immune responses to selected LTBI antigens were associated with subsequent reduced risk of progression to TB. METHODS: We conducted a population-based cohort study in East Greenland (2012-2014) including individuals aged 5-31years. A personal identifier allowed follow-up in national registers including the TB notification register. Mtb infection was defined by a positive Quantiferon test. Immune responses to LTBI antigens were assessed by whole blood antigen stimulation and interferon gamma measurement. RESULTS: Among 978 participants, 67 previously had TB. LTBI antigen (Rv1284, Rv2659, Rv2660c) immune response prevalence was 18%, 50%, 2% among Mtb-infected and 7%, 40%, 4% among non-infected (Quantiferon negative) participants. Among 911 participants without prior notified TB, 31 were notified with TB during study follow-up. Immune responses to LTBI antigens were not associated with reduced risk of subsequent TB; Rv1284 HR 0.92 (95%CI 0.28-3.04), Rv2659 HR 1.05 (95%CI 0.51-2.13), Rv2660c HR 3.06 (95%CI 0.70-13.37). CONCLUSION: In this large population-based study, human immune responses to selected LTBI antigens were not found to be strongly associated with reduced risk of subsequent TB.

Testing the H56 Vaccine Delivered in 4 Different Adjuvants as a BCG-Booster in a Non-Human Primate Model of Tuberculosis.

The search for new and improved tuberculosis (TB) vaccines has focused on IFN-γ both for selecting antigens and for evaluating vaccine delivery strategies. The essential role of IFN-γ in endogenous host protection is well established, but it is still uncertain whether this also holds true for vaccine protection. Here we evaluate the H56 fusion protein vaccine as a BCG booster in a non-human primate (NHP) model of TB that closely recapitulates human TB pathogenesis. To date, only a handful of novel adjuvants have been tested in the NHP model of TB, and therefore we administered H56 in 3 novel cationic liposome adjuvants of increasing immunogenicity (CAF01, CAF04, CAF05) and compared them to H56 in the IC31® adjuvant previously reported to promote protection in this model. The individual clinical parameters monitored during infection (weight, ESR, X-ray) all correlated with survival, and boosting BCG with H56 in all adjuvants resulted in better survival rates compared to BCG alone. The adjuvants promoted IFN-γ-responses of increasing intensity as measured by ELISPOT in the peripheral blood, but the level of vaccine-specific IFN-γ production did not correlate with or predict disease outcome. This study's main outcome underscores the importance of the choice of adjuvant for TB subunit vaccines, and secondly it highlights the need for better correlates of protection in preclinical models of TB.

The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING-Dependent Induction of Type I Interferons.

The cationic polysaccharide chitosan is an attractive candidate adjuvant capable of driving potent cell-mediated immunity, but the mechanism by which it acts is not clear. We show that chitosan promotes dendritic cell maturation by inducing type I interferons (IFNs) and enhances antigen-specific T helper 1 (Th1) responses in a type I IFN receptor-dependent manner. The induction of type I IFNs, IFN-stimulated genes and dendritic cell maturation by chitosan required the cytoplasmic DNA sensor cGAS and STING, implicating this pathway in dendritic cell activation. Additionally, this process was dependent on mitochondrial reactive oxygen species and the presence of cytoplasmic DNA. Chitosan-mediated enhancement of antigen specific Th1 and immunoglobulin G2c responses following vaccination was dependent on both cGAS and STING. These findings demonstrate that a cationic polymer can engage the STING-cGAS pathway to trigger innate and adaptive immune responses.

Different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens.

The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza, and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use.

The Memory Immune Response to Tuberculosis.

Immunological memory is a central feature of the adaptive immune system and a prerequisite for generating effective vaccines. Understanding long-term memory responses to Mycobacterium tuberculosis will thus provide us with valuable insights that can guide us in the search for a novel vaccine against tuberculosis (TB). For many years, triggering CD4 T cells and, in particular, those secreting interferon-γ has been the goal of most TB vaccine research, and numerous data from animals and humans support the key role of this subset in protective immunity. More recently, we have learned that the memory response required for effective control of M. tuberculosis is much more complex, probably involving several phenotypically different CD4 T cell subsets as well as other cell types that are yet to be defined. Herein, we describe recent insights into memory immunity to TB in the context of both animal models and the human infection. With the increasing amount of data generated from clinical testing of novel TB vaccines, we also summarize recent knowledge of vaccine-induced memory immunity.

Novel adjuvant formulations for delivery of anti-tuberculosis vaccine candidates.

There is an urgent need for a new and improved vaccine against tuberculosis for controlling this disease that continues to pose a global health threat. The current research strategy is to replace the present BCG vaccine or boost BCG-immunity with subunit vaccines such as viral vectored- or protein-based vaccines. The use of recombinant proteins holds a number of production advantages including ease of scalability, but requires an adjuvant inducing cell-mediated immune responses. A number of promising novel adjuvant formulations have recently been designed and show evidence of induction of cellular immune responses in humans. A common trait of effective TB adjuvants including those already in current clinical testing is a two-component approach combining a delivery system with an appropriate immunomodulator. This review summarizes the status of current TB adjuvant research with a focus on the division of labor between delivery systems and immunomodulators.

Protein energy malnutrition during vaccination has limited influence on vaccine efficacy but abolishes immunity if administered during Mycobacterium tuberculosis infection.

Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including tuberculosis (TB), but it is not clear how PEM influences vaccine-promoted immunity to TB. We demonstrate that PEM during low-level steady-state TB infection in a mouse model results in rapid relapse of Mycobacterium tuberculosis, as well as increased pathology, in both Mycobacterium bovis BCG-vaccinated and unvaccinated animals. PEM did not change the overall numbers of CD4 T cells in BCG-vaccinated animals but resulted in an almost complete loss of antigen-specific cytokine production. Furthermore, there was a change in cytokine expression characterized by a gradual loss of multifunctional antigen-specific CD4 T cells and an increased proportion of effector cells expressing gamma interferon and tumor necrosis factor alpha (IFN-γ(+) TNF-α(+) and IFN-γ(+) cells). PEM during M. tuberculosis infection completely blocked the protection afforded by the H56-CAF01 subunit vaccine, and this was associated with a very substantial loss of the interleukin-2-positive memory CD4 T cells promoted by this vaccine. Similarly, PEM during the vaccination phase markedly reduced the H56-CAF01 vaccine response, influencing all cytokine-producing CD4 T cell subsets, with the exception of CD4 T cells positive for TNF-α only. Importantly, this impairment was reversible and resupplementation of protein during infection rescued both the vaccine-promoted T cell response and the protective effect of the vaccine against M. tuberculosis infection.

Aluminium hydroxide potentiates a protective Th1 biased immune response against polio virus that allows for dose sparing in mice and rats.

BACKGROUND: The development of new low cost inactivated polio virus based vaccines (IPV) is a high priority and will be essential for the complete eradication of polio. Since the aluminium hydroxide adjuvant is widely used in humans we tested this adjuvant with IPV in two models. Our objective was twofold; to examine the IPV dose sparing effect of aluminium hydroxide and how the adjuvant effect of aluminium hydroxide affected the immunity induced by IPV. METHODS: Mice and rats were immunized with IPV formulated with Aluminium hydroxide and subjected to immunological analyses and serum polio virus neutralization titer determination. RESULTS: Addition of aluminium hydroxide to IPV led to a ten times dose sparing effect compared to IPV alone, measured by virus neutralization titers in serum. Aluminium hydroxide changed the kinetics of the response against IPV leading to a faster and stronger response, which due to IPV induced immune dominance was characterized as a strong Th1-biased cellular/humoral immune response. CONCLUSIONS: The IPV-aluminium hydroxide formulation constitutes a promising vaccine capable of generating strong Th1 immunity against infection with all three serotypes. A phase I/II clinical study was recently initiated.