Comparison of media for a human peripheral blood mononuclear cell-based in vitro vaccine evaluation system.

Purpose: Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Materials and Methods: Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. Results: With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Conclusion: Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

Prolonging the delivery of influenza virus vaccine improves the quantity and quality of the induced immune responses in mice.

Introduction: Influenza vaccines play a vital role in protecting individuals from influenza virus infection and severe illness. However, current influenza vaccines have suboptimal efficacy, which is further reduced in cases where the vaccine strains do not match the circulating strains. One strategy to enhance the efficacy of influenza vaccines is by extended antigen delivery, thereby mimicking the antigen kinetics of a natural infection. Prolonging antigen availability was shown to quantitatively enhance influenza virus-specific immune responses but how it affects the quality of the induced immune response is unknown. Therefore, the current study aimed to investigate whether prolongation of the delivery of influenza vaccine improves the quality of the induced immune responses over that induced by prime-boost immunization. Methods: Mice were given daily doses of whole inactivated influenza virus vaccine for periods of 14, 21, or 28 days; the control group received prime-boost immunization with a 28 days interval. Results: Our data show that the highest levels of cellular and humoral immune responses were induced by 28 days of extended antigen delivery, followed by 21, and 14 days of delivery, and prime-boost immunization. Moreover, prolonging vaccine delivery also improved the quality of the induced antibody response, as indicated by higher level of high avidity antibodies, a balanced IgG subclass profile, and a higher level of cross-reactive antibodies. Conclusions: Our findings contribute to a better understanding of the immune response to influenza vaccination and have important implications for the design and development of future slow-release influenza vaccines.

The evolution of humoral immune responses to past and novel influenza virus strains gives evidence for antigenic seniority.

The high genetic and antigenic variability of influenza virus and the repeated exposures of individuals to the virus over time account for the human immune responses toward this pathogen to continuously evolve during the lifespan of an individual. Influenza-specific immune memory to past strains has been shown to affect the immune responses to subsequent influenza strains and in turn to be changed itself through the new virus encounter. However, exactly how and to what extent this happens remains unclear. Here we studied pre-existing immunity against influenza A virus (IAV) by assessing IAV binding (IgG), neutralizing, and neuraminidase-specific antibodies to 5 different IAV strains in 180 subjects from 3 different age cohorts, adolescents, adults, and elderly, over a 5-year time span. In each age cohort, the highest neutralizing antibody titers were seen for a virus strain that circulated early in their life but the highest increase in titer was found for the most recent virus strains. In contrast, the highest IgG titers were seen against recent virus strains but the biggest increase in titer occurred against older strains. Significant increases in neutralizing antibody titers against a newly encountered virus strain were observed in all age cohorts demonstrating that pre-existing immunity did not hamper antibody induction. Our results indicate that the evolution of influenza-specific humoral immunity differs for rather cross-reactive virus-binding antibodies and more strain-specific neutralizing antibodies. Nevertheless, in general, our observations lend support to the antigenic seniority theory according to which the antibody response to influenza is broadened with each virus encounter, with the earliest encountered strain taking in the most senior and thus dominant position.

Characterization of humoral immune responses and degree of protection induced by influenza vaccine in cotton rats: Effects of low vaccine dose and single vs booster vaccination.

INTRODUCTION: Cotton rats are a suitable model for the study of influenza disease symptoms and responses to influenza vaccination. We have previously shown that two immunizations with 15 µg whole inactivated virus (WIV) influenza vaccine could completely protect animals from infection with the H1N1pdm09 virus. METHODS: To further explore the cotton rat model, we here investigated the protective potential of a single intramuscular immunization and of prime/boost intramuscular immunizations with a low amount of antigen. RESULTS: A single intramuscular immunization with doses more than or equal to 0.5 µg WIV reliably evoked antibody responses and doses more than or equal to 1 µg protected the animals from virus replication in the lungs and from severe weight loss. However, clinical symptoms like an increased respiration rate were still apparent. Administration of a booster dose significantly increased the humoral immune responses but did not or only moderately improved protection from clinical symptoms. CONCLUSION: Our data suggest that complete and partial protection by influenza vaccines can be mimicked in cotton rats by using specific vaccination regimens.

Cross-Protective Potential and Protection-Relevant Immune Mechanisms of Whole Inactivated Influenza Virus Vaccines Are Determined by Adjuvants and Route of Immunization.

Adjuvanted whole inactivated virus (WIV) influenza vaccines show promise as broadly protective influenza vaccine candidates. Using WIV as basis we assessed the relative efficacy of different adjuvants by carrying out a head-to-head comparison of the liposome-based adjuvants CAF01 and CAF09 and the protein-based adjuvants CTA1-DD and CTA1-3M2e-DD and evaluated whether one or more of the adjuvants could induce broadly protective immunity. Mice were immunized with WIV prepared from A/Puerto Rico/8/34 (H1N1) virus intramuscularly with or without CAF01 or intranasally with or without CAF09, CTA1-DD, or CTA1-3M2e-DD, followed by challenge with homologous, heterologous or heterosubtypic virus. In general, intranasal immunizations were significantly more effective than intramuscular immunizations in inducing virus-specific serum-IgG, mucosal-IgA, and splenic IFNγ-producing CD4 T cells. Intranasal immunizations with adjuvanted vaccines afforded strong cross-protection with milder clinical symptoms and better control of virus load in lungs. Mechanistic studies indicated that non-neutralizing IgG antibodies and CD4 T cells were responsible for the improved cross-protection while IgA antibodies were dispensable. The role of CD4 T cells was particularly pronounced for CTA1-3M2e-DD adjuvanted vaccine as evidenced by CD4 T cell-dependent reduction of lung virus titers and clinical symptoms. Thus, intranasally administered WIV in combination with effective mucosal adjuvants appears to be a promising broadly protective influenza vaccine candidate.

Inactivated or damaged? Comparing the effect of inactivation methods on influenza virions to optimize vaccine production.

The vast majority of commercially available inactivated influenza vaccines are produced from egg-grown or cell-grown live influenza virus. The first step in the production process is virus inactivation with ß-propiolactone (BPL) or formaldehyde (FA). Recommendations for production of inactivated vaccines merely define the maximal concentration for both reagents, leaving the optimization of the process to the manufacturers. We assessed the effect of inactivation with BPL and FA on 5 different influenza virus strains. The properties of the viral formulation, such as successful inactivation, preservation of hemagglutinin (HA) binding ability, fusion capacity and the potential to stimulate a Toll-like receptor 7 (TLR7) reporter cell line were then assessed and compared to the properties of the untreated virus. Inactivation with BPL resulted in undetectable infectivity levels, while FA-treated virus retained very low infectious titers. Hemagglutination and fusion ability were highly affected by those treatments that conferred higher inactivation, with BPL-treated virus binding and fusing at a lower degree compared to FA-inactivated samples. On the other hand, BPL-inactivated virus induced higher levels of activation of TLR7 than FA-inactivated virus. The alterations caused by BPL or FA treatments were virus strain dependent. This data shows that the inactivation procedures should be tailored on the virus strain, and that many other elements beside the concentration of the inactivating agent, such as incubation time and temperature, buffer and virus concentration, have to be defined to achieve a functional product.

Cross-Protective Immune Responses Induced by Sequential Influenza Virus Infection and by Sequential Vaccination With Inactivated Influenza Vaccines.

Sequential infection with antigenically distinct influenza viruses induces cross-protective immune responses against heterologous virus strains in animal models. Here we investigated whether sequential immunization with antigenically distinct influenza vaccines can also provide cross-protection. To this end, we compared immune responses and protective potential against challenge with A(H1N1)pdm09 in mice infected sequentially with seasonal A(H1N1) virus followed by A(H3N2) virus or immunized sequentially with whole inactivated virus (WIV) or subunit (SU) vaccine derived from these viruses. Sequential infection provided solid cross-protection against A(H1N1)pdm09 infection while sequential vaccination with WIV, though not capable of preventing weight loss upon infection completely, protected the mice from reaching the humane endpoint. In contrast, sequential SU vaccination did not prevent rapid and extensive weight loss. Protection correlated with levels of cross-reactive but non-neutralizing antibodies of the IgG2a subclass, general increase of memory T cells and induction of influenza-specific CD4+ and CD8+ T cells. Adoptive serum transfer experiments revealed that despite lacking neutralizing activity, serum antibodies induced by sequential infection protected mice from weight loss and vigorous virus growth in the lungs upon A(H1N1)pdm09 virus challenge. Antibodies induced by WIV vaccination alleviated symptoms but could not control virus growth in the lung. Depletion of T cells prior to challenge revealed that CD8+ T cells, but not CD4+ T cells, contributed to cross-protection. These results imply that sequential immunization with WIV but not SU derived from antigenically distinct viruses could alleviate the severity of infection caused by a pandemic and may improve protection to unpredictable seasonal infection.

Pulmonary delivery of influenza vaccine formulations in cotton rats: site of deposition plays a minor role in the protective efficacy against clinical isolate of H1N1pdm virus.

Administration of influenza vaccines to the lungs could be an attractive alternative to conventional parenteral administration. In this study, we investigated the deposition site of pulmonary delivered liquid and powder influenza vaccine formulations and its relation to their immunogenicity and protective efficacy. In vivo deposition studies in cotton rats revealed that, the powder formulation was mainly deposited in the trachea ( ∼ 65%) whereas the liquid was homogenously distributed throughout the lungs ( ∼ 96%). In addition, only 60% of the antigen in the powder formulation was deposited in the respiratory tract with respect to the liquid formulation. Immunogenicity studies showed that pulmonary delivered liquid and powder influenza formulations induced robust systemic and mucosal immune responses (significantly higher by liquids than by powders). When challenged with a clinical isolate of homologous H1N1pdm virus, all animals pulmonary administered with placebo had detectable virus in their lungs one day post challenge. In contrast, none of the vaccinated animals had detectable lung virus titers, except for two out of eight animals from the powder immunized group. Also, pulmonary vaccinated animals showed no or little signs of infection like increase in breathing frequency or weight loss upon challenge as compared to animals from the negative control group. In conclusion, immune responses induced by liquid formulation were significantly higher than responses induced by powder formulation, but the overall protective efficacy of both formulations was comparable. Thus, pulmonary immunization is capable of inducing protective immunity and the site of antigen deposition seems to be of minor relevance in inducing protection.

Monophosphoryl Lipid A-Adjuvanted Virosomes with Ni-Chelating Lipids for Attachment of Conserved Viral Proteins as Cross-Protective Influenza Vaccine.

Induction of CD8+ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross-protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen-presenting cells (APCs) and get access to the MHC class I antigen processing pathway. To facilitate the generation of NP-specific CTL immunity the authors develop a novel influenza vaccine consisting of virosomes with the Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal-ion-chelating lipid DOGS-NTA-Ni incorporated in the membrane. In vitro, virosomes with incorporated MPLA induce stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS-NTA-Ni show high conjugation efficacy for his-tagged proteins and facilitate efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA-adjuvanted virosomes with attached NP results in priming of NP-specific CTLs while MPLA-adjuvanted virosomes with admixed NP are inefficient in priming CTLs. Both vaccines induce equally high titers of NP-specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP are protected from severe weight loss. Yet, unexpectedly, they show more weight loss and more severe disease symptoms than mice immunized with MPLA-virosomes without NP. Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen-specific antibody responses in vivo. However, for protection from influenza infection NP-specific immunity appears not to be advantageous.

Distinctive Responses in an In Vitro Human Dendritic Cell-Based System upon Stimulation with Different Influenza Vaccine Formulations.

Vaccine development relies on testing vaccine candidates in animal models. However, results from animals cannot always be translated to humans. Alternative ways to screen vaccine candidates before clinical trials are therefore desirable. Dendritic cells (DCs) are the main orchestrators of the immune system and the link between innate and adaptive responses. Their activation by vaccines is an essential step in vaccine-induced immune responses. We have systematically evaluated the suitability of two different human DC-based systems, namely the DC-cell line MUTZ-3 and primary monocyte-derived DCs (Mo-DCs) to screen immunopotentiating properties of vaccine candidates. Two different influenza vaccine formulations, whole inactivated virus (WIV) and subunit (SU), were used as model antigens as they represent a high immunogenic and low immunogenic vaccine, respectively. MUTZ-3 cells were restricted in their ability to respond to different stimuli. In contrast, Mo-DCs readily responded to WIV and SU in a vaccine-specific way. WIV stimulation elicited a more vigorous induction of activation markers, immune response-related genes and secretion of cytokines involved in antiviral responses than the SU vaccine. Furthermore, Mo-DCs differentiated from freshly isolated and freeze/thawed peripheral blood mononuclear cells (PBMCs) showed a similar capacity to respond to different vaccines. Taken together, we identified human PBMC-derived Mo-DCs as a suitable platform to evaluate vaccine-induced immune responses. Importantly, we show that fresh and frozen PBMCs can be used indistinctly, which strongly facilitates the routine use of this system. In vitro vaccine pre-screening using human Mo-DCs is thus a promising approach for evaluating the immunopotentiating capacities of new vaccine formulations that have not yet been tested in humans.

Adjuvantation of Pulmonary-Administered Influenza Vaccine with GPI-0100 Primarily Stimulates Antibody Production and Memory B Cell Proliferation.

Adjuvants are key components in vaccines, they help in reducing the required antigen dose but also modulate the phenotype of the induced immune response. We previously showed that GPI-0100, a saponin-derived adjuvant, enhances antigen-specific mucosal and systemic antibody responses to influenza subunit and whole inactivated influenza virus (WIV) vaccine administered via the pulmonary route. However, the impact of the GPI-0100 dose on immune stimulation and the immune mechanisms stimulated by GPI-0100 along with antigen are poorly understood. Therefore, in this study we immunized C57BL/6 mice via the pulmonary route with vaccine consisting of WIV combined with increasing amounts of GPI-0100, formulated as a dry powder. Adjuvantation of WIV enhanced influenza-specific mucosal and systemic immune responses, with intermediate doses of 5 and 7.5 µg GPI-0100 being most effective. The predominant antibody subtype induced by GPI-0100-adjuvanted vaccine was IgG1. Compared to non-adjuvanted vaccine, GPI-0100-adjuvanted WIV vaccine gave rise to higher numbers of antigen-specific IgA- but not IgG-producing B cells in the lungs along with better mucosal and systemic memory B cell responses. The GPI-0100 dose was negatively correlated with the number of influenza-specific IFNγ- and IL17-producing T cells and positively correlated with the number of IL4-producing T cells observed after immunization and challenge. Overall, our results show that adjuvantation of pulmonary-delivered WIV with GPI-0100 mostly affects B cell responses and effectively induces B cell memory.

Innate responses induced by whole inactivated virus or subunit influenza vaccines in cultured dendritic cells correlate with immune responses in vivo.

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates.

Comparison of adjuvants for a spray freeze-dried whole inactivated virus influenza vaccine for pulmonary administration.

Stable vaccines administered to the lungs by inhalation could circumvent many of the problems associated with current immunizations against respiratory infections. We earlier provided proof of concept in mice that pulmonary delivered whole inactivated virus (WIV) influenza vaccine formulated as a stable dry powder effectively elicits influenza-specific antibodies in lung and serum. Yet, mucosal IgA, considered particularly important for protection at the site of virus entry, was poorly induced. Here we investigate the suitability of various Toll-like receptor (TLR) ligands and the saponin-derived compound GPI-0100 to serve as adjuvant for influenza vaccine administered to the lungs as dry powder. The TLR ligands palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN) as well as GPI-0100 tolerated the process of spray freeze-drying well. While Pam3CSK4 had no effect on systemic antibody titers, all the other adjuvants significantly increased influenza-specific serum and lung IgG titers. Yet, only GPI-0100 also enhanced mucosal IgA titers. Moreover, only GPI-0100-adjuvanted WIV provided partial protection against heterologous virus challenge. Pulmonary immunization with GPI-0100-adjuvanted vaccine did not induce an overt inflammatory response since influx of neutrophils and production of inflammatory cytokines were moderate and transient and lung histology was normal. Our results indicate that a GPI-0100-adjuvanted dry powder influenza vaccine is a safe and effective alternative to current parenteral vaccines.

Influenza virosomes supplemented with GPI-0100 adjuvant: a potent vaccine formulation for antigen dose sparing.

Adjuvants can stimulate vaccine-induced immune responses and can contribute decisively to antigen dose sparing when vaccine antigen production is limited, as for example during a pandemic influenza outbreak. We earlier showed that GPI-0100, a semi-synthetic saponin derivative with amphiphilic structure, significantly stimulates the immunogenicity and protective efficacy of influenza subunit vaccine administered via a systemic route. Here, we evaluated the adjuvant effect of GPI-0100 on a virosomal influenza vaccine formulation. In contrast to influenza subunit vaccine adjuvanted with GPI-0100, virosomal vaccine supplemented with the same dose of GPI-0100 provided full protection of mice against infection at the extremely low antigen dose of 2 × 8 ng hemagglutinin. Overall, adjuvanted virosomes elicited higher antibody and T-cell responses than did adjuvanted subunit vaccine. The enhanced immunogenicity of the GPI-0100-adjuvanted virosomes, particularly at low antigen doses, is possibly due to a physical association of the amphiphilic adjuvant with the virosomal membrane. These results show that a combination of GPI-0100 and a virosomal influenza vaccine formulation is highly immunogenic and allows the use of very low antigen doses without compromising the protective potential of the vaccine.

Evaluation of mucosal and systemic immune responses elicited by GPI-0100- adjuvanted influenza vaccine delivered by different immunization strategies.

Vaccines for protection against respiratory infections should optimally induce a mucosal immune response in the respiratory tract in addition to a systemic immune response. However, current parenteral immunization modalities generally fail to induce mucosal immunity, while mucosal vaccine delivery often results in poor systemic immunity. In order to find an immunization strategy which satisfies the need for induction of both mucosal and systemic immunity, we compared local and systemic immune responses elicited by two mucosal immunizations, given either by the intranasal (IN) or the intrapulmonary (IPL) route, with responses elicited by a mucosal prime followed by a systemic boost immunization. The study was conducted in BALB/c mice and the vaccine formulation was an influenza subunit vaccine supplemented with GPI-0100, a saponin-derived adjuvant. While optimal mucosal antibody titers were obtained after two intrapulmonary vaccinations, optimal systemic antibody responses were achieved by intranasal prime followed by intramuscular boost. The latter strategy also resulted in the best T cell response, yet, it was ineffective in inducing nose or lung IgA. Successful induction of secretory IgA, IgG and T cell responses was only achieved with prime-boost strategies involving intrapulmonary immunization and was optimal when both immunizations were given via the intrapulmonary route. Our results underline that immunization via the lungs is particularly effective for priming as well as boosting of local and systemic immune responses.

Enhancement of the immunogenicity and protective efficacy of a mucosal influenza subunit vaccine by the saponin adjuvant GPI-0100.

Identification of safe and effective adjuvants remains an urgent need for the development of inactivated influenza vaccines for mucosal administration. Here, we used a murine challenge model to evaluate the adjuvant activity of GPI-0100, a saponin-derived adjuvant, on influenza subunit vaccine administered via the intranasal or the intrapulmonary route. Balb/c mice were immunized with 1 µg A/PR/8 (H1N1) subunit antigen alone or in combination with varying doses of GPI-0100. The addition of GPI-0100 was required for induction of mucosal and systemic antibody responses to intranasally administered influenza vaccine and significantly enhanced the immunogenicity of vaccine administered via the intrapulmonary route. Remarkably, GPI-0100-adjuvanted influenza vaccine given at a low dose of 2×1 µg either in the nares or directly into the lungs provided complete protection against homologous influenza virus infection.

The effect of pre-existing immunity on the capacity of influenza virosomes to induce cytotoxic T lymphocyte activity.

Protein antigens encapsulated in virosomes generated from influenza virus can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. In the present study we determined, in a murine model system, whether pre-existing immunity against influenza virus hampers the induction of a CTL response. CTL induction was only slightly reduced by pre-injection of influenza virus-specific antibodies or pre-exposure to influenza virus. Both pretreatments resulted in the same level of reduction, suggesting that virus-specific antibodies rather than T cell responses account for the reduction. Furthermore, a booster immunization enhanced CTL activation, indicating that virosome-specific immunity induced by a prime immunization does not hamper the booster effect. In conclusion, CTL induction against virosome-encapsulated protein antigens is not significantly inhibited by pre-existing humoral or cellular immunity against influenza virus.

Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia.

It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV-16 E7 specific immune responses in patients with different grades of cervical intra-epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T-cell responses were measured by IFN-gamma ELISPOT analysis, after stimulation with recombinant HPV-16 E7 protein. As expected, HPV-16 E7 specific IFN-gamma T cell responses were more frequent in HPV-16 DNA positive patients compared with that in HPV-16 DNA negative patients (39/50 vs. 16/36, (p=0.006, chi2 test). After invasive procedures, a small number of HPV-16 DNA positive CIN patients, but a considerable proportion of HPV-16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV-16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4+ and CD8+ T cells showed E7 specific IFN-gamma production upon in-vitro stimulation. Our study shows that invasive procedures may enhance HPV-specific cell-mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV-specific immune responses is used as intermediate end-point.

A virosomal immunization strategy against cervical cancer and pre-malignant cervical disease.

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7-specific CTL response as assessed by 51Cr release measurements and MHC tetramer staining of spleen cells. Immunization with E7-containing virosomes also resulted in E7-specific antibody responses. In tumour challenge experiments, immunization of mice with E7-containing virosomes prevented tumour outgrowth in >70% of the animals. Thus, influenza-derived virosomes with encapsulated HPV E7 protein antigen act as an excellent vaccine delivery system for induction of cellular immunity against HPV-transformed cells and represent a promising immunotherapeutic vaccine for the treatment of (precursor lesions of) cervical cancer.

Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity.

Induction of CTL responses against protein antigens is an important aim in vaccine development. In this paper we present fusion-active virosomes as a vaccine delivery system capable of efficient induction of CTL responses in vivo. Virosomes are reconstituted viral membranes, which do not contain the genetic material of the virus they are derived from. Foreign macromolecules, including protein antigens, can be encapsulated in virosomes during the reconstitution process. Functionally reconstituted virosomes retain the cell binding and fusion characteristics of the native virus. Thus, upon uptake by cells through receptor-mediated endocytosis, virosomes will deliver their content to the cell cytosol. In a previous study, we demonstrated that protein antigens delivered in this manner to dendritic cells are efficiently processed for both MHC class I and class II presentation. Here, we studied in vivo induction of cellular immune responses against virosome-encapsulated ovalbumin (OVA) in mice. As little as 0.75 microg OVA delivered by fusion-active virosomes was sufficient to induce a powerful class I MHC-restricted CTL response. All immunization routes that were used (i.m., i.p. and s.c.) resulted in efficient induction of CTL activity. The CTLs induced were cytotoxic in a standard 51Cr-release assay and produced IFNgamma in response to OVA peptide. Thus, virosomes represent an ideal antigen delivery system for induction of cellular immunity against encapsulated protein antigens.