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Newcastle disease (ND) is a highly fatal, infectious, viral disease, and despite immunization with live and inactivated vaccines, the disease is still endemic, causing heavy morbidity and mortality leading to huge economic losses to the poultry industry in Pakistan. Therefore, the present study was aimed for the first time in the country at using novel virosomal technology to develop the ND vaccine using an indigenous highly virulent strain of the virus. ND virosome was prepared using Triton X-100, and SM2 Bio-Beads were used to remove the detergent and reconstitute the viral membrane into virosome. Confirmation was done by transmission electron microscopy and protein analysis by SDS-PAGE. In vitro cell adhesion property was observed by incorporating green fluorescent protein (GFP), producing plasmid into virosome and in vitro cell culture assay. Sterility, safety, and stability of the vaccine were tested before in vivo evaluation of immunogenicity and challenge protection study in commercial broiler. The virosome vaccine was administered (30 μg/bird) at days 7 and 14 through the intranasal route in comparison with commercially available live and inactivated ND vaccines. Results revealed significantly high ( p < 0.05 ) and clinically protective hemagglutination inhibition (HI) antibody titers at 7, 14, 21, and 28 days postimmunization with the virosome vaccine in comparison to the negative control. The GMTs were comparable to live and inactivated vaccines with nonsignificant ( p > 0.05 ) differences throughout the experiment. Antibody levels increased in all vaccinated groups gradually from the 7th day and were maximum at 28th-day postvaccination. In the virosome-administered group, GMT was 83.18 and 77.62 at 21st and 28th-days postvaccination, respectively. Challenge revealed 100%, 90%, and 80% protection in virosome, live, and inactivated vaccinated groups, respectively. Under given experimental conditions, we can conclude that ND virosome vaccine prepared from the indigenous virus was found to be safe and immunogenic.

Current SARS-CoV-2 vaccines are effective, but long-term protection is threatened by the emergence of virus variants. We generated a virosome vaccine containing the Beta spike protein and compared its immunogenicity in mice to a virosome vaccine containing the original Wuhan spike. Two administrations of the virosomes induced potent SARS-CoV-2 neutralizing antibodies in both vaccine groups. The level of autologous neutralization in Beta-vaccinated mice was similar to the level of autologous neutralization in Wuhan-vaccinated mice. However, heterologous neutralization to the Wuhan strain in Beta-vaccinated mice was 4.7-fold lower than autologous neutralization, whereas heterologous neutralization to the Beta strain in Wuhan-vaccinated mice was reduced by only 1.9-fold compared to autologous neutralization levels. In addition, neutralizing activity against the D614G, Alpha and Delta variants was also significantly lower after Beta spike vaccination than after Wuhan spike vaccination. Our results show that Beta spike vaccination induces inferior neutralization breadth. These results are informative for programs aimed to develop broadly active SARS-CoV-2 vaccines.

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5μg, 7.5μg, or 30μg), or a non-adjuvanted vaccine (30μg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5μg and 30μg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30μg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection.

Influenza A virus is one of the most important zoonotic pathogens that can cause severe symptoms and has the potential to cause high number of deaths and great economic loss. Vaccination is still the best option to prevent influenza virus infection. Different types of influenza vaccines, including live attenuated virus vaccines, inactivated whole virus vaccines, virosome vaccines, split-virion vaccines and subunit vaccines have been developed. However, they have several limitations, such as the relatively high manufacturing cost and long production time, moderate efficacy of some of the vaccines in certain populations, and lack of cross-reactivity. These are some of the problems that need to be solved. Here, we summarized recent advances in the development and application of different types of influenza vaccines, including the recent development of viral vectored influenza vaccines. We also described the construction of other vaccines that are based on recombinant influenza viruses as viral vectors. Information provided in this review article might lead to the development of safe and highly effective novel influenza vaccines.

Characterization of virosomes, in late stage preclinical development as vaccines for Respiratory Syncytial Virus (RSV), with a membrane-incorporated synthetic monophosphoryl lipid A, 3D-PHADA (R) adjuvant.Virosomes were initially formed by contacting a lipid film containing 3D-PHADA (R) with viral membranes solubilized with the short chain phospholipid DCPC, followed by dialysis, later by adding solubilized 3D-PHAD to viral membranes, or to preformed virosomes from DMSO.Virosomes formed from lipid films contained the membrane glycoproteins G and F, at similar F to G ratios but lower concentrations than in virus, and the added lipids, but only a fraction of the 3D-PHADA (R). By single particle tracking (SPT), the virosome size distribution resembled that seen by cryo-electron microscopy, but dynamic light scattering showed much larger particles. These differences were caused by small virosome aggregates. Measured by SPT, virosomes were stable for 300 days. 3DPHAD A (R) incorporation in virosomes could be enhanced by providing the adjuvant from DCPC solubilized stock, but also by adding DMSO dissolved adjuvant to pre-formed virosomes. Virosomes with 0.1 mg/mg of 3D-PHADA (R)/viral protein from DMSO induced antibody titers similar to those by virosomes containing 0.2 mg/mg of DCPC-solubilized 3D-PHADA (R).Stable 3D-PHADA (R) adjuvanted RSV virosomes can be formulated.

Background: The pediatric dose of the virosomal hepatitis A vaccine Epaxal (R), Epaxal (R) Junior, is safe and immunogenic in children from 1 to 17 years of age. The present study investigated the long-term immunogenicity of Epaxal (R) Junior. The standard doses of Epaxal (R) and aluminum-adsorbed hepatitis A vaccine (Havrix (R) Junior) were used as comparators. Methods: A total of 271 children who had completed a 0/6-month immunization schedule (priming and booster dose) participated in this follow-up study. Anti-hepatitis A virus (HAV) antibody levels were measured using a microparticle enzyme immunoassay (HAVAB 2.0 Quantitative; Abbott Diagnostics, Wiesbaden, Germany) starting at 18 months following the second dose, and then yearly until 66 months (ie, 5.5 years) after the second dose. Results: All subjects tested at Month 66 still had protective anti-HAV antibodies (>= 10 mIU/mL). Antibody titers were generally lower in subjects 1-7 years old than in subjects 8-17 years old and higher in females 11-17 years old than in males 11-17 years old. In addition, an age-dependent decay was observed, that is, antibody decreased more rapidly in younger than in older children. Conclusions: Vaccination of children with two doses of Epaxal (R) Junior confers a real-time protection of at least 5.5 years. This protection is estimated to last approximately 25 years. Younger children showed lower antibody titers and a faster antibody decline than older children. Additional follow-up studies are needed beyond 5.5 years to further assess the longterm immunogenicity of Epaxal (R) Junior.

Immunosenescence contributes to increased incidence and severity of many infections in old age and is responsible for impaired immunogenicity and efficacy of vaccines. Adjuvants are one strategy to enhance immunogenicity of vaccines. The oil-in-water emulsions MF59TM and AS03, as well as a virosomal vaccine have been licensed in seasonal or pandemic influenza vaccines and are/were used successfully in the elderly. AS01, a liposome-based adjuvant comprising two immunostimulants has recently been approved in a recombinant protein vaccine for older adults, which showed very high efficacy against herpes zoster in clinical trials. Several adjuvants for use in the older population are in clinical and preclinical development and will hopefully improve vaccines for this age group in the future.

Evidence from both animal and human viral diseases indicate that cytotoxic T lymphocytes (CTL) are crucial in antiviral defense. However, a major problem to generate cytotoxic immunity is that in vivo exogenous antigens are usually presented via MHC class II pathway and normally fail to induce CTL. The aim of this study is to describe a novel non-live prototype vaccine based on immunopotentiating reconstituted influenza virosomes (IRIV) as vehicles to deliver HLA-A*0201-restricted hepatitis C virus (HCV) peptides (core 35-44 and 131-140) into the cytoplasm of at least three different target cell types [including T2, a transporter associated with antigen processing (TAP)-deficient cell line] resulting in MHC class I peptide presentation and lysis by peptide-specific CTL lines. Comparison of kinetics and analysis of the influence of peptide-stripping and Brefeldin A (BFA) reveal that there exists an endogenous, TAP-independent and BFA-sensitive pathway for virosomally delivered peptides. Moreover, virosomes containing influenza matrix peptide 58-66 can efficiently re-stimulate in vivo primed CTL and, importantly, IRIV containing HCV core peptides can even prime CTL from peripheral blood mononuclear cells of HCV(-) healthy blood donors in vitro. The fact that in vitro primed CTL are also able to specifically lyse target cells infected with recombinant vaccinia virus encoding the HCV core protein is of great importance for future studies based on in vivo mouse models. One of the most evident advantages of the virosomes in vivo will be their capability to protect the incorporated peptide from a large variety of degrading proteases.

Aim: To clarify the immunogenicity and safety of influenza vaccine in patients with end-stage kidney disease (ESKD) on dialysis or who have received a kidney transplant. Methods: Sixty adolescents and young adults with ESKD (25 on hemodialysis and 35 kidney transplant recipients) were randomized 1:1 to receive a traditional trivalent split virion vaccine (TIIV) or a virosome-adjuvanted trivalent inactivated influenza vaccine (VATIIV). The immunogenicity and safety of the two vaccines was evaluated and compared with the findings observed in 30 healthy subjects of similar age and gender distribution who received TIIV. Results: The results indicate that the immune response of the patients to TIIV and VATIIV were similar. The administered vaccines were safe and well tolerated, and no advantage was found with the use of VATIIV. Conclusion: Given the potential clinical relevance of influenza in patients with ESKD, these findings support the official recommendation that they should receive annual influenza vacci...

T cellular responses play a significant role in mediating protective immune responses against influenza in humans. In the current study, we evaluated the ability of a candidate virosomal H5N1 vaccine adjuvanted with Matrix M(TM) to induce CD4(+) and CD8(+) T cell responses in a phase 1 clinical trial. We vaccinated 60 healthy adult volunteers (at days 0 and 21) with 30 μg haemagglutinin (HA) alone or 1.5, 7.5, or 30 μg HA formulated with Matrix M(TM). To evaluate the T cellular responses, lymphocytes were stimulated in vitro with homologous (A/Vietnam/1194/2004 [H5N1]) and heterologous H5N1 (A/Anhui/1/05 or A/Bar-headed Goose/Qinghai/1A/05) antigens. The antigen-specific cytokine responses were measured by intracellular cytokine staining and by multiplex (Luminex) assays. An increase in CD4(+) Th1 and Th2 cytokines was detected 21 days after the first vaccine dose. No increase in Th cytokine responses was observed after the second dose, although it is possible that the cytokine levels peaked earlier than sampling point at day 42. Formulation with the Matrix M(TM) adjuvant augmented both the homologous and cross-reactive cytokine response. Antigen-specific CD8(+) T cell responses were detected only in a few vaccinated individuals. The concentrations of Th1 and to a lesser extent, Th2 cytokines at 21 days post-vaccination correlated moderately with subsequent days 35 and 180 serological responses as measured by the microneutralisation, haemagglutination inhibition, and single radial hemolysis assays. Results presented here show that the virosomal H5N1 vaccine induced balanced Th1/Th2 cytokine responses and that Matrix M(TM) is a promising adjuvant for future development of candidate pandemic influenza vaccines.

Acute disseminated encephalomyelitis (ADEM) is an inflammatory, usually monophasic, immune mediate, demyelinating disease of the central nervous system which involves the white matter. ADEM is more frequent in children and usually occurs after viral infections, but may follow vaccinations, bacterial infections, or may occur without previous events. Only 5% of cases of ADEM are preceded by vaccination within one month prior to symptoms onset. The diagnosis of ADEM requires both multifocal involvement and encephalopathy and specific demyelinating lesions of white matter. Overall prognosis of ADEM patients is often favorable, with full recovery reported in 23% to 100% of patients from pediatric cohorts, and more severe outcome in adult patients. We describe the first case of ADEM occurred few days after administration of virosomal seasonal influenza vaccine. The patient, a 59-year-old caucasic man with unremarkable past medical history presented at admission decreased alertness, 10 days after flu vaccination. During the 2 days following hospitalization, his clinical conditions deteriorated with drowsiness and fever until coma. The magnetic resonance imaging of the brain showed multiple and symmetrical white matter lesions in both cerebellar and cerebral hemispheres, suggesting demyelinating disease with inflammatory activity, compatible with ADEM. The patient was treated with high dose of steroids and intravenous immunoglobulin with relevant sequelae and severe neurological outcomes.

Background Non-replicating respiratory syncytial virus (RSV) vaccine candidates could potentially prime for enhanced respiratory disease (ERD) due to a T-cell-mediated immunopathology, following RSV infection. Vaccines with built-in immune response modifiers, such as Toll-like receptor (TLR) ligands, may avoid such aberrant imprinting of the immune system. Methods We developed reconstituted RSV envelopes (virosomes) with incorporated TLR4 ligand, monophosphoryl lipid A (RSV-MPLA virosomes). Immune responses and lung pathology after vaccination and challenge were investigated in ERD-prone cotton rats and compared with responses induced by live virus and formaldehyde-inactivated vaccine (FI-RSV), a known cause of ERD upon RSV challenge. Results Vaccination with RSV-MPLA virosomes induced higher levels of virus-neutralizing antibodies than FI-RSV or live virus infection and provided protection against infection. FI-RSV, but not RSV-MPLA virosomes, primed for increases in expression of Th2 cytokines IL-4, IL-5, IL-13, and Th1 cytokine IL-1b, 6 hour–5 days after infection. By contrast, RSV-MPLA virosomes induced IFN-γ transcripts to similar levels as induced by live virus. Animals vaccinated with FI-RSV, but not RSV-MPLA virosomes showed alveolitis, with prominent neutrophil influx and peribronchiolar and perivascular infiltrates. Conclusion These results show that RSV-MPLA virosomes represent a safe and immunogenic vaccine candidate that warrants evaluation in a clinical setting.

Annual vaccination is the main mean of preventing influenza in the elderly. In order to evaluate the effectiveness of the adjuvanted seasonal influenza vaccines available in Italy in preventing hospitalization for influenza and pneumonia, a matched case-control study was performed in elderly subjects during the 2010-2011 season in Genoa (Italy). Cases and controls were matched in a 1:1 ratio according to gender, age, socio-economic status and type of influenza vaccine. Vaccine effectiveness was calculated as IVE = [(1-OR)x100] and crude odds ratios were estimated through conditional logistic regression models. Adjusted odds ratios were estimated through multivariable logistic models. In the study area, influenza activity was moderate in the 2010-2011 season, with optimal matching between circulating viruses and vaccine strains. We recruited 187 case-control pairs; 46.5% of cases and 79.1% of controls had been vaccinated. The adjuvanted influenza vaccines (Fluad (®) considered together with Inflexal V (®) ) were associated with a significant reduction in the risk of hospitalization, their effectiveness being 94.8% (CI 77.1-98.8). Adjusted vaccine effectiveness was 95.2% (CI 62.8-99.4) and 87.8 (CI 0.0-98.9) for Inflexal V (®) and Fluad (®) , respectively. Both adjuvanted vaccines proved effective, although the results displayed statistical significance only for Inflexal V (®) (p = 0.004), while for Fluad (®) statistical significance was not reached (p = 0.09). Our study is the first to provide information on the effectiveness of Inflexal V (®) in terms of reducing hospitalizations for influenza or pneumonia in the elderly, and demonstrates that this vaccine yields a high degree of protection and that its use would generate considerable saving for the National Health Service.

Objective We performed a Phase 1 randomized, observer-blinded, placebo-controlled trial to evaluate the safety and immunogenicity of a recombinant respiratory syncytial virus (RSV) fusion (F) protein nanoparticle vaccine. Methods Six formulations with (5, 15, 30 and 60 μg) and without (30 and 60 μg) aluminum phosphate (AdjuPhos) were administered intramuscularly on day 0 and 30 in a dose escalating fashion to healthy adults 18–49 years of age. Solicited and unsolicited events were collected through day 210. Immunogenicity measures taken at day 0, 30 and 60 included RSV A and B microneutralization, anti-F IgG, antigenic site II peptide and palivizumab competitive antibodies. Results The vaccine was well-tolerated, with no evident dose-related toxicity or attributable SAEs. At day 60 both RSV A and B microneutralization was significantly increased in vaccinees versus placebo. Across all vaccinees there was a 7- to 19-fold increase in the anti-F IgG and a 7- to 24-fold increase in the antigenic site II binding and palivizumab competitive antibodies. Conclusions The RSV F nanoparticle vaccine candidate was well tolerated without dose-related increases in adverse events. Measures of immunity indicate that neutralization, anti-RSV F IgG titers and palivizumab competing antibodies were induced at levels that have been associated with decreased risk of hospitalization. NCT01290419 .

Background The aim of this open-label, active-controlled, parallel group, phase 2 follow-up study was to assess the long-term immunogenicity of Epaxal Junior, the pediatric dose of an aluminum-free virosomal inactivated hepatitis A virus (HAV) vaccine, in children receiving routine childhood vaccines (RCV). Methods Healthy children (12-15 months old, ≥8 kg weight) were randomized (1:1:1) to group A: Epaxal Junior + RCV (day 1); group B: Epaxal Junior (day 1) + RCV (day 29) and group C: Havrix 720 + RCV (day 1). All 3 groups received 2 doses of HAV vaccines 6 months apart. Children who completed the primary study were followed up from 18 months to 7.5 years post booster. Results Of 291/327 randomized children who had completed the primary study, 157 were followed for the 7.5-year analysis (group A: 50; group B: 54; and group C: 53). Of these, 152 children had protective levels of anti-HAV antibodies [≥10 mIU/mL; 98% (group A); 96.3% (group B); 96.2% (group C)]. Anti-HAV geometric mean concentrations were similar in groups A and B at all the time points (1.5-, 2.5-, 3.5-, 5.25- and 7.5-year time point) but slightly lower in group C. Predictions of the median duration of persistence of seroprotective antibody levels, using the linear mixed model were similar in all groups: (group A: 19.1 years, group B: 18.7 years, group C: 17.3 years). Conclusions Immunization with Epaxal Junior administered with RCVs at 12 months elicited protective response beyond 7.5 years in almost all children. Assessing the kinetic of anti-HAV antibody titers decline over time, the moment to reach antibody concentrations below the accepted protective level may occur earlier than previously estimated.

Although respiratory syncytial virus (RSV) is a significant human pathogen, no RSV vaccines are available. We have reported that a virus-like particle (VLP) RSV vaccine candidate stimulated, in mice, robust, protective anti-RSV glycoprotein T H 1 biased immune responses without enhanced respiratory disease upon RSV challenge. We report here an analysis of long-term responses to these VLPs. BALB/c mice immunized, without adjuvant, with VLPs or with infectious RSV generated anti-F and anti-G protein serum antibody responses that were stable over 14 months. Neutralizing antibody titers stimulated by VLPs were robust and durable for 14 months, whereas those of RSV-immunized animals declined significantly by 3 months. F protein-specific antibody-secreting cells were detected in the bone marrows of VLP-immunized mice but not in the marrows of RSV-immunized mice. Adoptive transfer of enriched splenic B cells from VLP-immunized mice into immunodeficient rag −/− mice resulted in anti-F and anti-G protein serum IgG antibody responses, in recipient mice, that were protective upon RSV challenge. In contrast, transfer of splenic B cells from RSV-immunized mice produced no detectable serum antibody in the recipients, nor could these mice inhibit RSV replication upon virus challenge. Immunization with VLPs stimulated the formation of germinal center GL7 + B cells in normal mice. VLP immunization of TCR βδ −/− T-cell-deficient mice did not induce anti-RSV IgG antibodies, results consistent with T-cell-dependent immune responses. These results demonstrate that VLPs are effective in stimulating long-lived RSV-specific, T-cell-dependent neutralizing antibody-secreting cells and RSV-specific memory responses.

The discovery of broadly neutralizing antibodies that recognize highly conserved epitopes in the membrane-proximal region of influenza virus hemagglutinin (HA) has revitalized efforts to develop a universal influenza virus vaccine. This effort will likely require novel immunogens that contain these epitopes but lack the variable and immunodominant epitopes located in the globular head of HA. As a first step toward developing such an immunogen, we investigated whether the 20-residue A-helix of the HA2 chain that forms the major component of the epitope of broadly neutralizing antibodies CR6261, F10, and others is sufficient by itself to elicit antibodies with similarly broad antiviral activity. Here, we report the multivalent display of the A-helix on icosahedral virus-like particles (VLPs) derived from the capsid of Flock House virus. Mice immunized with VLPs displaying 180 copies/particle of the A-helix produced antibodies that recognized trimeric HA and the elicited antibodies had binding characteristics similar to those of CR6261 and F10: they recognized multiple HA subtypes from group 1 but not from group 2. However, the anti-A-helix antibodies did not neutralize influenza virus. These results indicate that further engineering of the transplanted peptide is required and that display of additional regions of the epitope may be necessary to achieve protection.

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.

Virus-like particles (VLPs) can be generated from Chikungunya virus (CHIKV), but different strains yield variable quantities of particles. Here, we define the genetic basis for these differences and show that amino acid 234 in E2 substantially affects VLP production. This site is located within the acid-sensitive region (ASR) known to initiate a major conformational change in E1/E2. Selected other mutations in the ASR, or changes in pH, also increased VLP yield. These results demonstrate that the ASR of E2 plays an important role in regulating particle generation.

The exploratory immunogenicity objective of this analysis was to characterize the titer of vaccine human papillomavirus (HPV)-type immunoglobulins in both peripartum maternal blood and the cord blood of infants born to women who received blinded therapy. Data were derived from a randomized, placebo-controlled, double-blind safety, immunogenicity, and efficacy study (protocol 019; NCT00090220). This study enrolled 3,819 women between the ages of 24 and 45 years from 38 international study sites between 18 June 2004 and 30 April 2005. Data in the current analysis are from subjects enrolled in Philippines and Thailand. For each of HPV types 6, 11, 16, and 18, maternal anti-HPV was found in cord blood samples. Furthermore, HPV titers in cord blood samples were highly positively correlated with maternal HPV titers. Additionally, there were instances when anti-HPV antibodies were no longer detectable in maternal serum samples and yet were detected in matched cord blood samples. These results demonstrate that quadrivalent HPV (qHPV) vaccine-induced antibodies cross the placenta and could potentially provide some benefit against vaccine-type HPV infection and related diseases such as recurrent respiratory papillomatosis.

CIA05 is a toll-like receptor (TLR) 4 agonist derived from an Escherichia coli lipopolysaccharide (LPS) mutant and has been shown to have potential as a vaccine adjuvant. In this study, we investigated the immunopotentiating activity of the adjuvant system CIA06, which is comprised of CIA05 and aluminum hydroxide (alum), when used with the human papillomavirus (HPV) L1 virus-like particles (VLPs) vaccine. BALB/c mice were immunized intramuscularly three times at 2-week intervals with HPV16 L1 VLPs alone or in the presence of various combinations of CIA05 and alum, and the immune responses were assessed. We found that the combination of CIA05 and alum at a ratio of 1:50 (designated CIA06B) yielded the highest immune response in terms of serum anti-HPV L1 VLP IgG antibody titers, splenocyte interferon (IFN)-γ secretion, and antigen-specific memory B cell responses. The immunogenicity of the CIA06B-adjuvanted HPV16/18 L1 VLP vaccine was compared with that of the currently licensed HPV vaccine Cervarix™. The CIA06B-adjuvanted vaccine was similar to Cervarix™ with regard to eliciting serum antigen-specific IgG antibodies and virus-neutralizing antibodies but more effective at inducing splenic cytokine production and memory B cells. We also observed that the antigen-specific IgG antibody titers, splenic IFN-γ secretion and memory B cells induced by the CIA06B-adjuvanted HPV vaccine remained high up to 24 weeks post-immunization. Based on these data, we concluded that CIA06B may have potential as an adjuvant in a potent prophylactic vaccine against HPV infection.

The extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region ofSalmonella entericaserovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.

Background. Vaccination is the best measure to protect the population against a potential influenza H5N1 pandemic, but 2 doses of vaccine are needed to elicit protective immune responses. An immunological marker for H5N1 vaccine effectiveness is needed for early identification of the best vaccine candidate. Methods. We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with Matrix M. Sixty adult volunteers were vaccinated intramuscularly with 2 doses of either 30 μg hemagglutinin (HA) alone or with 1.5, 7.5, or 30 μg HA and Matrix M adjuvant (50 μg). The humoral response was measured by the hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) assays, and the CD4 + T-helper 1 (Th1)–cell response was measured by intracellular staining for the cytokines interleukin 2, interferon γ, and tumor necrosis factor α. Results. The adjuvanted vaccine effectively induced CD4 + Th1-cell responses, and the frequency of influenzaspecific Th1 cells after the first vaccine dose predicted subsequent HI, MN, and SRH seroprotective responses after the second vaccination. Conclusions. These results support early identification of Th1-cell responses as a predictive biomarker for an efficient vaccine response, which could have great implications for early identification of persons with low or no response to vaccine when evaluating future pandemic influenza vaccines. Clinical Trials Registration. NCT00868218.

Several studies have described the production of influenza virus-like particles (VLP) using a variety of platform systems. These VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins and have been proposed as vaccine candidates for both seasonal and pandemic influenza. Although still in the early stages of development and evaluation as influenza vaccines, influenza VLPs have a variety of other valuable uses such as examining and understanding correlates of protection against influenza and investigating virus-cell interactions. The most common production system for influenza VLPs is the baculovirus-insect cell expression which has several attractive features including the ease in which new gene combinations can be constructed, the immunogenicity elicited and protection afforded by the produced VLPs, and the scalability offered by the system. However, there are differences between the influenza VLPs produced by baculovirus expression systems in insect cells and the influenza viruses produced for use as current vaccines or the virus produced during a productive clinical infection. We describe here the development of a modified vaccinia virus Ankara (MVA) system to generate mammalian influenza VLPs containing influenza H5N1 proteins. The MVA vector system is flexible for manipulating and generating various VLP constructs, expresses high level of influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins, and can be scaled up to produce VLPs in quantities sufficient for in vivo studies. We show that mammalian VLPs are generated from recombinant MVA vectors expressing H5N1 HA alone, but that increased VLP production can be achieved if NA is co-expressed. These mammalian H5N1 influenza VLPs have properties in common with live virus, as shown by electron microscopy analysis, their ability to hemagglutinate red blood cells, express neuraminidase activity, and to bind influenza specific antibodies. Importantly, these VLPs are able to elicit a protective immune response in a mouse challenge model, suggesting their utility in dissecting the correlates of immunity in such models. Mammalian derived VLPs may also provide a useful tool for studying virus-cell interactions and may have potential for development as pandemic vaccines.

To determine whether immunotherapy with HPV6 L1 virus like particles (VLPs) without adjuvant (VLP immunotherapy) reduces recurrence of genital warts following destructive therapy.A randomized placebo controlled blinded study of treatment of recurrent genital warts amenable to destructive therapy, conducted independently in Australia and China.Patients received conventional destructive therapy of all evident warts together with intramuscular administration of 1 µg, 5 µg or 25 µg of VLP immunotherapy, or of placebo immunotherapy (0.9% NaCl), as immunotherapy at week 0 and week 4. Primary outcome, assessed at week 8, was recurrence of visible warts.Of 33 protocol compliant Brisbane recipients of placebo immunotherapy, 11 were disease free at two months, and a further 9 demonstrated reduction of50% in total wart area. Wart area reduction following destructive treatment correlated with prior duration of disease. Among 102 protocol compliant Brisbane recipients of VLP immunotherapy, disease reduction was significantly greater than among the placebo immunotherapy (50% ± s.e.m. 7%) recipients for subjects receiving 5 µg or 25 µg of VLP immunotherapy/dose (71% ± s.e.m. 7%) but not for those receiving 1 µg VLP immunotherapy/dose (42% ± 7%). Of 52 protocol compliant placebo immunotherapy recipients in Wenzhou, 37 were disease free at two months, and a further 8 had50% disease reduction. Prior disease duration was much shorter in Wenzhou subject (8.1 ± 1.1 mo) than in Brisbane subjects (53.7 ± 5.5 mo). No significant reduction in mean wart area was observed for the 168 Wenzhou protocol compliant subjects who also received VLP immunotherapy.This study confirms the findings in a previous open label trial that administration of VLP immunotherapy may assist in clearance of recurrent genital warts in patients for whom destructive therapy is unsuccessful and that unsuccessful destructive therapy is more common with increasing prior disease duration.

Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 “Consensus” VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH) 3 ) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.

Current infectious bursal disease virus (IBDV) vaccines suffer from maternal antibody interference and mimotope vaccines might be an alternative. Previously we demonstrated an IBDV VP2 five-mimotope polypeptide, 5EPIS, elicited protective immunity in chickens. In the current study, the 5epis gene was inserted into a plasmid carrying human hepatitis B virus core protein (HBc) gene at its major immunodominant region site. The recombinant gene was efficiently expressed in Escherichia coli to produce chimeric protein HBc-5EPIS which self-assembles to virus-like particles (VLP). Two-week old specific-pathogen-free chickens were immunized intramuscularly with HBc-5EPIS VLP or 5EPIS polypeptide without adjuvant (50 μg/injection) on day 0, 7, 14 and 21. Anti-5EPIS antibody was first detected on day 7 and day 21 in HBc-5EPIS and 5EPIS groups, respectively; on day 28, anti-5EPIS titers reached 12,800 or 1600 by ELISA, and 3200 or 800 by virus neutralization assay in HBc-5EPIS and 5EPIS groups, respectively. No anti-5EPIS antibody was detected in the buffer control group throughout the experiment. Challenge on day 28 with a virulent IBDV strain (GX8/99) resulted in 100%, 40.0% and 26.7% survival for chickens immunized with HBc-5EPIS, 5EPIS and buffer, respectively. These data suggest epitope presentation on chimeric VLP is a promising approach for improving mimotope vaccines for IBDV.

We assessed the immunogenicity of the paediatric dose of Epaxal(®) (0.25 mL) and the degrees of seroprotection achieved with the standard dose (0.5 mL) of Epaxal(®) or a dose of Havrix(®) Junior, in children in an open, randomised, controlled, multi-centre, parallel-group study conducted at 2 Chilean study centres. 360 healthy children and adolescents 12 months to

Transmission of pathogenic avian influenza viruses (AIV) from wild birds to domestic poultry and humans is continuing in multiple countries around the world. In preparation for a potential AIV pandemic, multiple vaccine candidates are under development. In the case of H5N1 AIV, a clear shift in transmission from clade 1 to clade 2 viruses occurred in recent years. The virus-like particle (VLP) represents an economical approach to pandemic vaccine development. In the current study, we evaluated the humoral immune response in humans vaccinated with H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine manufactured in Sf9 insect cells. The VLPs were comprised of the influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins. In an FDA-approved phase I/II human clinical study, two doses of H5N1 VLPs at 15, 45, or 90 μg HA/dose resulted in seroconversion and production of functional antibodies. Moreover, cross-reactivity against other clade 2 subtypes was demonstrated using virus neutralization assays. H5N1 whole-genome fragment phage display libraries (GFPDL) were used to elucidate the antibody epitope repertoire in postvaccination human sera. Diverse epitopes in HA1/HA2 and NA were recognized by postvaccination sera from the two high-dose groups, including large segments spanning the HA1 receptor binding domain. Importantly, the vaccine elicited sera that preferentially bound to an oligomeric form of recombinant HA1 compared with monomeric HA1. The oligomeric/monomeric HA1 binding ratios of the sera correlated with the virus neutralizing titers. Additionally, the two high-dose VLP vaccine groups generated NA-inhibiting antibodies that were associated with binding to a C-terminal epitope close to the sialic acid binding site. These findings represent the first report describing the quality of the antibody responses in humans following AIV VLP immunization and support further development of such vaccines against emerging influenza virus strains.

Virus-like particles hold great promise for the development of effective and affordable vaccines. Indeed, virus-like particles are suitable for presentation and efficient delivery of linear as well as conformational antigens to antigen-presenting cells. This will ultimately result in optimal B-cell activation and cross-presentation with both MHC class I and II molecules to prime CD4(+) T-helper as well as CD8(+) cytotoxic T cells. This article provides an update on the development and use of virus-like particles as vaccine approaches for infectious diseases and cancer.

Highlights ► The safety and immunogenicity of one or two doses of a recombinant A (H1N1) 2009 influenza VLP vaccine was evaluated in a two-stage, Phase 2, randomized, double-blind, placebo-controlled study conducted during the H1N1 2009 pandemic in Mexico. ► The H1N1 2009 VLP vaccine was safe and well-tolerated with no vaccine-related serious adverse events. ► The VLP vaccine groups demonstrated robust HAI immune responses after a single vaccination, which were statistically higher compared to placebo., Virus-like particles (VLPs) can be rapidly developed from influenza virus genetic sequences in order to supply vaccine after the onset of a pandemic. The safety and immunogenicity of one or two doses of a recombinant A (H1N1) 2009 influenza VLP vaccine was evaluated in a two-stage, Phase 2, randomized, double-blind, placebo-controlled study conducted in 4563 healthy adults, 18–64 years of age, during the H1N1 2009 pandemic in Mexico. In Part A, 1013 subjects were randomized into four treatment groups (5 μg, 15 μg, or 45 μg hemagglutinin [HA] VLP vaccine or placebo) and vaccinated 21 days apart, with sera collected on Days 1, 14 and 36 for hemagglutination inhibition (HAI) testing. After review of safety and immunogenicity data from Part A, additional subjects were immunized with a single dose of 15 μg VLP vaccine (N = 2537) or placebo (N = 1011) and assessed for safety in Part B. Results showed the H1N1 2009 VLP vaccine was safe and well-tolerated. Systemic solicited events were similar between placebo and VLP vaccinated groups with no vaccine-related serious adverse events. Dose response trends for solicited local adverse events were observed, with higher incidences of local pain, swelling, tenderness, and redness reported in the higher VLP dose groups (15 μg and 45 μg) compared to the placebo and 5 μg VLP groups following both vaccinations. Although the majority of local AEs were mild in severity, a dose trend in events of moderate or greater severity was also noted for these solicited events. The VLP vaccine groups demonstrated robust HAI immune responses after a single vaccination, with high rates of seroprotection (≥40 HAI titer) in 82–92% of all subjects and in 64–85% of subjects who were seronegative at the time of immunization. HAI geometric mean titers (GMTs), geometric mean ratios (GMRs) and seroconversion rates were also all statistically higher in the VLP groups compared to placebo for both post-baseline time points. Based on these data, additional clinical trials are in development to evaluate influenza vaccine candidate antigens manufactured using Spodoptera frugiperda (Sf9)/baculovirus-based VLP technology.

Studies on a platform technology able to deliver low-cost viral capsomeres and virus-like particles are described. The technology involves expression of the VP1 structural protein from murine polyomavirus (MuPyV) in Escherichia coli, followed by purification using scaleable units and optional cell-free VLP assembly. Two insertion sites on the surface of MuPyV VP1 are exploited for the presentation of the M2e antigen from influenza and the J8 peptide from Group A Streptococcus (GAS). Results from testing on mice following subcutaneous administration demonstrate that VLPs are self adjuvating, that adding adjuvant to VLPs provides no significant benefit in terms of antibody titre, and that adjuvanted capsomeres induce an antibody titre comparable to VLPs but superior to unadjuvanted capsomere formulations. Antibodies raised against GAS J8 peptide following immunization with chimeric J8-VP1 VLPs are bactericidal against a GAS reference strain. E. coli is easily and widely cultivated, and well understood, and delivers unparalleled volumetric productivity in industrial bioreactors. Indeed, recent results demonstrate that MuPyV VP1 can be produced in bioreactors at multi-gram-per-litre levels. The platform technology described here therefore has the potential to deliver safe and efficacious vaccine, quickly and cost effectively, at distributed manufacturing sites including those in less developed countries. Additionally, the unique advantages of VLPs including their stability on freeze drying, and the potential for intradermal and intranasal administration, suggest this technology may be suited to numerous diseases where adequate response requires large-scale and low-cost vaccine manufacture, in a way that is rapidly adaptable to temporal or geographical variation in pathogen molecular composition.

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal–epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.

Influenza virus-like particles (VLPs) are effective vaccines against influenza infection, which can be produced either in insect cells by recombinant baculovirus (BV) infection or in mammalian cells by DNA plasmid transfection. However, VLPs produced from baculovirus/insect cells are difficult to purify due to baculovirus contamination; VLPs produced by plasmid transfection are limited by scale-up capability. In this study, a BacMam BV, in which three CMV-promoters drive the hemagglutinin, neuraminidase, and matrix of influenza virus was constructed. This baculovirus can deliver these genes into mammalian cells/hosts and subsequently influenza VLPs can be produced and secreted from transduced cells. Transduction conditions were optimized and influenza VLPs were purified from transduced 293T cells. Mice were vaccinated with BV transduction-produced VLPs, plasmid transfection-produced VLPs, and BacMam BV. Two vaccinations of each vaccine induced high hemagglutination-inhibition (HAI) titers and prevented influenza virus infection. In contrast, following a single vaccination, all mice vaccinated with each vaccine had significantly lower lung viral titers compared to unvaccinated mice. Remarkably, mice vaccinated with a single dose of BV transduction-produced VLPs survived challenge, whereas mice vaccinated with one dose of BacMam BV- or plasmid transfection-produced VLPs had 60-80% survival. This finding is particularly significant for producing easily purified VLPs. The BacMam system is an alternative strategy for VLP production, which is easy to scale up and purify. Besides, BacMam BV can be used as a gene delivery vector to produce VLPs in vivo, to stimulate immune responses.

Human immunodeficiency virus infection is a worldwide health problem and a protective vaccine is desperately needed to control the AIDS pandemics. To address this concern, we previously constructed single-cycle replicable (SCR) HIV-1 virions, which completely maintained the antigenic structures of HIV-1. Herein, to optimize a vaccination strategy, we studied the immunogenicity of produced SCR virions and adjuvant-formulated HIV-1 virus-like particles (VLPs) in homologous and heterologous prime-boosting regimens. Accordingly, BALB/c mice received three doses of immunogens in 3-week intervals and their immune responses were evaluated using ELISA, cytokine and IFN-γ ELISpot assays. These analyses not only indicated the superiority of SCR prime-VLP boosting for strong induction of specific IFN-γ producing cells, but also showed the capability of this strategy over the others for better stimulation of humoral response, which was evidenced with the detection of highest titer of total IgG against HIV ENV glycoprotein. Furthermore, determination of IgG subclasses and IFN-γ/IL4 secretion ratio in cultured splenocytes demonstrated the efficient augmentation of mixed responses with the dominancy of Th1 immunity following SCR/VLP immunization strategy. Our results additionally pointed towards the applicability of Montanide ISA 720 + CpG as a potent Th1-directing adjuvant mixture. Overall, this study suggests SCR prime-VLP boosting as a promising approach in HIV vaccine development.

The norovirus P particle is an octahedral nanoparticle formed by 24 copies of the protrusion (P) domain of the norovirus capsid protein. This P particle is easily produced in Escherichia coli , extremely stable, and highly immunogenic. There are three surface loops per P domain, making a total of 72 loops per particle, and these are potential sites for foreign antigen presentation for immune enhancement. To prove this concept, a small peptide (His tag, 7 amino acids [aa]) and a large antigen (rotavirus VP8, 159 aa) were inserted into one of the loops. Neither insertion affects P particle formation, while both antigens were presented well on the P particle surface. The immune-enhancement effect of the P particle was demonstrated by significantly increased antibody titers induced by the P particle-presented antigens compared to the titers induced by free antigens. In addition, the measured neutralization antibody titers and levels of protection against rotavirus shedding in mice immunized with the VP8 chimeric P particles were significantly higher than those of mice immunized with the free VP8 antigen. Sera from P particle-VP8 chimera-vaccinated animals also blocked norovirus virus-like particle (VLP) binding to the histo-blood group antigen (HBGA) receptors. From these data, the P particle appears to be an excellent vaccine platform for antigen presentation. The readily available three surface loops and the great capacity for foreign antigen insertion make this platform attractive for wide application in vaccine development and antibody production. The P particle-VP8 chimeras may serve as a dual vaccine against both rotavirus and norovirus.

Human norovirus (HuNoV) is a major causative agent of food-borne gastroenteritis worldwide. Currently, there are no vaccines or effective therapeutic interventions for this virus. Development of an attenuated vaccine for HuNoV has been hampered by the inability to grow the virus in cell culture. Thus, a vector-based vaccine may be ideal. In this study, we constructed a recombinant vesicular stomatitis virus (rVSV-VP1) expressing VP1, the major capsid protein of HuNoV. Expression of the capsid protein by VSV resulted in the formation of HuNoV virus-like particles (VLPs) that are morphologically and antigenically similar to native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 through intranasal and oral routes stimulated a significantly stronger humoral and cellular immune response than baculovirus-expressed VLP vaccination. Moreover, we demonstrated that mice inoculated with rVSV-VP1 triggered a comparable level of fecal and vaginal IgA antibody. Taken together, the VSV recombinant system not only provides a new approach to generate HuNoV VLPs in vitro but also a new avenue for the development of vectored vaccines against norovirus and other noncultivable viruses.

We describe here a single plasmid DNA immunogen representing the broadest antigen repertoire among HIV vaccine candidates. This pDNA was "ANTIGENeered" for the regulated expression of thirteen complete and two non-functional HIV protein antigens. These proteins self assemble into complex virus-like particles (VLP(+)). Multiple irreversible safety features were introduced by genetic modifications including the complete impairment of integration, reverse transcription, the function of Nef and the 3'LTR. Epitope analysis predicted that the pDNA-derived protein repertoire can potentially present over 3000 T cell epitopes. However, the expressed antigens have different potential to induce HIV-specific CD4(+) and CD8(+) T cells supporting our hypothesis that HIV vaccines should contain all possible regulatory and structural proteins. This immunogen is the active pharmaceutical ingredient of DermaVir, a therapeutic vaccine product candidate that recently successfully completed Phase II clinical trials and meets the safety, immunogenicity and cost requirements of an HIV vaccine.

Norwalk virus (NV) is an enteric pathogen from the genus Norovirus and a major cause of nonbacterial gastroenteritis in humans. NV virus-like particles (VLPs) are known to elicit systemic and mucosal immune responses when delivered nasally; however, the correlates of immune protection are unknown, and codelivery with a safe and immunogenic mucosal adjuvant may enhance protective anti-NV immune responses. Resiquimod (R848), an imidazoquinoline-based Toll-like receptor 7 and/or 8 (TLR7/8) agonist, is being evaluated as an adjuvant in FDA-approved clinical vaccine trials. As such, we evaluated the adjuvant activity of two imidazoquinoline-based TLR7 and TLR7/8 agonists when codelivered intranasally with plant-derived NV VLPs. We also compared the activity of these agonists to the gold standard mucosal adjuvant, cholera toxin (CT). Our results indicate that codelivery with the TLR7 agonist, gardiquimod (GARD), induces NV VLP-specific serum IgG and IgG isotype responses and mucosal IgA responses in the gastrointestinal, respiratory, and reproductive tracts that are superior to those induced by R848 and comparable to those induced by the mucosal adjuvant CT. This study supports the continued investigation of GARD as a mucosal adjuvant for NV VLPs and possible use for other VLP-based vaccines for which immune responses at distal mucosal sites (e.g., respiratory and reproductive tracts) are desired.

To address the limitations of conventional influenza vaccine manufacturing and delivery, this study investigated administration of virus-like particle (VLP) influenza vaccine using a microneedle patch. The goal was to determine if skin immunization with influenza VLP vaccine using microneedles enables dose sparing. We found that low-dose influenza (A/PR/8/34 H1N1) VLP vaccination using microneedles was more immunogenic than low-dose intramuscular (IM) vaccination and similarly immunogenic as high-dose IM vaccination in a mouse model. With a 1 μg dose of vaccine, both routes showed similar immune responses and protective efficacy, with microneedle vaccination being more effective in inducing recall antibody responses in lungs and antibody secreting cells in bone marrow. With a low dose of vaccine (0.3 μg), microneedle vaccination induced significantly superior protective immunity, which included binding and functional antibodies as well as complete protection against a high dose lethal infection with A/PR/8/34 virus, whereas IM immunization provided only partial (40%) protection. Therefore, this study demonstrates that microneedle vaccination in the skin confers more effective protective immunity at a lower dose, thus providing vaccine dose-sparing effects.

Vaccines targeting the amyloid-β (Aβ) peptide have promise as immunotherapies for the treatment of Alzheimer disease (AD). Human trials of a first generation Aβ vaccine highlighted the need for a vaccine strategy that could consistently induce high-titer antibodies against Aβ without also inducing inflammatory auto-reactive T-cell responses. in this review, I will describe the use of virus-like particle (vLP) based vaccines against Aβ that can potentially satisfy these demands. vLPs can serve as highly multivalent platforms for the display of diverse antigens on their surfaces. vLP display markedly increases the immunogenicity of antigens, including self-antigens. vLP- based immunogens targeting Ab have been developed by several different groups, and have demonstrated effectiveness in animal models of AD. One vLP-based candidate vaccine for AD, CAD106, developed by Cytos Biotechnology and Novartis Pharmaceuticals, is currently in human clinical trials.

Background. The aim of this study was to investigate the immunogenicity, safety and tolerability of the 2009 A/H1N1 MF59-adjuvanted influenza vaccine, administered sequentially or simultaneously with the seasonal 2009–10 virosomal-adjuvanted influenza vaccine, to paediatric kidney transplant recipients. Methods. Thirty-two children and adolescents with transplanted kidneys and 32 age- and gender-matched healthy controls were randomized 1:1 to receive the pandemic vaccine upon enrolment and the seasonal vaccine 1 month later (16 transplant recipients and 16 healthy controls), or to receive the two vaccines simultaneously upon enrolment (16 transplant recipients and 16 healthy controls). Results. When the pandemic vaccine was administered sequentially to the seasonal vaccine, it was significantly less immunogenic in the patients than in the controls (P < 0.05); when it was administered together with the seasonal vaccine, the immune response of both patients (P < 0.05) and controls (P < 0.05) was significantly greater than when it was administered sequentially. Seroconversion rates and the geometric mean titres of all of the seasonal antigens were significantly lower in the patients, regardless of the type of vaccine administration (P < 0.05). Simultaneous administration was associated with a better immune response against A/H1N1 and A/H3N2 antigens in both patients and controls, and did not increase the mild local and systemic reactions. No impact on renal function was observed. Conclusions. Paediatric kidney transplant recipients have a lower immune response to the pandemic influenza A/H1N1 MF59-adjuvanted and seasonal virosomal-adjuvanted influenza vaccines than healthy controls. The simultaneous administration of the two vaccines seems to increase immune response to both pandemic and seasonal A/H1N1 and A/H3N2 antigens, and has the same safety profile as that of the pandemic vaccine administered sequentially to the seasonal vaccine.

New or improved vaccines against dengue virus types 1 to 4 (DENV1 to DENV4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are urgently required. The use of noninfectious subviral extracellular particles (EPs) is an inexpensive and safe strategy for the production of protein-based flavivirus vaccines. Although coexpression of premembrane (prM) and envelope (E) proteins has been demonstrated to produce EPs in mammalian cells, low yields have hindered their commercial application. Therefore, we used an insect cell expression system with Spodoptera frugiperda -derived Sf9 cells to investigate high-level production of DENV2 and JEV EPs. Sf9 cells transfected with the prM and E genes of DENV2 or JEV secreted corresponding viral antigens in a particulate form that were biochemically and biophysically equivalent to the authentic antigens obtained from infected C6/36 mosquito cells. Additionally, equivalent neutralizing antibody titers were induced in mice immunized either with EPs produced by transfected Sf9 cells or with EPs produced by transfected mammalian cells, in the context of coimmunization with a DNA vaccine that expresses EPs. Furthermore, the results of an enzyme-linked immunosorbent assay (ELISA) using an EP antigen derived from Sf9 cells correlated significantly with the results obtained by a neutralization test and an ELISA using an EP antigen derived from mammalian cells. Finally, Sf9 cells could produce 10- to 100-fold larger amounts of E antigen than mammalian cells. These results indicate the potential of Sf9 cells for high-level production of flavivirus protein vaccines and diagnostic antigens.

Few studies have examined the duration of protection following vaccination against hepatitis A virus (HAV) with currently licensed HAV vaccines. This study explored the long-term immunogenicity in individuals vaccinated with the virosomal hepatitis A virus, Epaxal®. Adult volunteers (N = 130) previously enrolled into four different studies between 1992 and 1994 and who had completed a 0/12-month immunization regimen (primary and booster dose) were asked to participate in this follow-up study. Yearly anti-HAV titers up to 6 years following booster vaccination, and then once 9–11 years after booster were measured using two assays, Enzygnost® and AxSYM® HAVAB 2.0. Based on the Enzygnost® assay, the seroprotection rate 9–11 years after booster was 100%, with a geometric mean concentration (GMC) of anti-HAV antibodies of 526 mIU/ml. Females had markedly higher GMCs than males (741 mIU/ml vs. 332 mIU/ml). Using an anti-HAV cut-off titer of ≥10 mIU/ml, a linear mixed mathematical model predicted a median duration of protection of 52.1 years. A duration of protection ≥35.7 years was predicted for 95% of subjects. A more stringent cut-off of ≥20 mIU/ml shortened the median predicted duration of protection to 45.0 years. In conclusion, a two-dose Epaxal® vaccination regimen confers in healthy adults a real-time protection of at least 9–11 years; this protection is predicted to last at least 30 years in over 95% of individuals. Further studies are necessary to assess the real duration of seroprotection and whether an additional booster is necessary later. J. Med. Virol. 82:1629–1634, 2010. 2010 Wiley-Liss, Inc.

This study evaluated the immunogenicity of a double dose of the seasonal virosomal-adjuvanted influenza vaccine (Inflexal V, Crucell, The Netherlands) in 65 previously unvaccinated children aged less than 3 years: 43 received double doses (two doses of 0.50 mL 4 weeks apart) and 22 standard doses (two doses of 0.25 mL 4 weeks apart). Both treatments evoked a response that satisfied the EMEA criteria for adequate immunogenicity for all three vaccine strains, but the double dose had a significantly greater effect on all of the studied parameters of humoral and cell-mediated immune response (p0.05). This result was achieved without any increase in the incidence of local and systemic adverse events. This means that doubling the dose of the virosomal-adjuvanted influenza vaccine (i.e. administering the same dose as that usually given to older children) effectively and safely increases the immune response to inactivated influenza vaccine in unprimed children aged less than 3 years.

Cervarix™ is a prophylactic human papillomavirus (HPV)-16/18 vaccine developed for the prevention of cervical cancer. The vaccine antigens are HPV-16 and HPV-18 L1 virus-like particles (VLPs) made from baculovirus expression vector system (BEVS)-produced HPV-16 and HPV-18 L1 proteins, respectively. In this study, we demonstrate that truncation of the nuclear targeting and DNA binding signals at the C-terminus of the HPV-16 and HPV-18 L1 proteins prevented intranuclear formation of the VLPs in the host cells and led to cytoplasmic localization of the L1 proteins as shown by in situ immunogold detection and electron microscopy. Following purification, these L1 proteins were able to form VLPs. The characteristics of these HPV-16 and HPV-18 L1 VLPs were studied using various physicochemical and immunological techniques. Amino acid analysis, SDS-PAGE and western blotting demonstrated the high purity of the L1 proteins and batch-to-batch consistency. The structure of the VLPs was shown to be similar to that reported for the native virions, as evaluated by microscopic observations, protein tomography and disc centrifugation experiments. The presence of important conformation-dependent neutralizing epitopes, such as U4, V5 and J4, was confirmed by ELISA and surface plasmon resonance. Structural robustness and consistency among batches was also observed by differential scanning calorimetry and electron microscopy. Moreover, adsorption to aluminum was shown not to impair VLP structure. In conclusion, the BEVS-produced HPV-16 and HPV-18 L1 VLPs display key structural and immunological features, which contribute to the efficacy of Cervarix™ vaccination.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

Infectious pancreatic necrosis virus (IPNV) is a major viral pathogen of salmonid fish and causes serious economic losses to salmonid aquaculture. Previously, we demonstrated that the IPNV capsid protein, VP2, expressed in yeast self-assembles into subviral particles (SVPs) and injection of these IPNV rVP2 SVPs into rainbow trout elicits an immune response. Immunized fish had reduced viral loads compared to unimmunized fish when challenged with IPNV. To evaluate the suitability of IPNV rVP2 SVPs for future development of multivalent vaccines, a linear epitope of a human oncogene, c-myc, was cloned into the IPNV rVP2 SVP backbone as a model epitope and expressed in yeast. Western blot analyses with anti-c-myc and anti-IPNV antibodies provided positive identification of both the c-myc and VP2 epitopes on the c-myc VP2 SVPs. Transmission electron microscopy of purified chimeric c-myc VP2 SVPs revealed the formation of approximately 20nm particles. Rainbow trout immunized with c-myc VP2 SVPs elicited both anti-c-myc and anti-IPNV immune responses. When immunized fish were challenged with IPNV, the viral load in the c-myc VP2 SVP immunized fish was significantly lower than the sham-vaccinated controls. The results indicate that IPNV rVP2 SVPs can tolerate the insertion of foreign epitopes without affecting either the antigenic potential of the epitopes of the backbone protein or the inserted foreign epitope. This opens the possibility of using the IPNV rVP2 SVP platform to express epitopes of other viruses, which could pave the way for development of multivalent subunit vaccines or novel marker vaccines.

We describe the safety of the human papillomavirus (HPV)-6/11/16/18 vaccine using updated clinical trial data (median follow-up time of 3.6 years) and summarize up to 3 years of post-licensure surveillance.In 5 clinical trials, 21,480 girls/women aged 9 to 26 years and boys aged 9 to 16 years receivedor=1 dose of HPV-6/11/16/18 vaccine or placebo. All serious and non-serious adverse experiences (AEs) and new medical conditions were recorded for the entire study period(s). As of June 2009,25 million doses of HPV-6/11/16/18 vaccine had been distributed in the United States with50 million doses globally. Post-licensure safety as summarized by the Centers for Disease Control and Prevention using the United States Vaccine Adverse Event Reporting System database is also reported.Eight subjects experienced a treatment-related serious AE (0.05% vaccine; 0.02% placebo). Of 18 deaths (0.1% vaccine; 0.1% placebo), all were considered unrelated to study treatment. New medical conditions which were potentially consistent with autoimmune phenomena were reported in 2.4% of both vaccine and placebo recipients. Pain, the most common injection-site AE, occurred more frequently with vaccine (81% vaccine; 75% placeboaluminum; 45% placebo-saline). No differences were seen in the incidence of the most common non-serious AEs-headache and pyrexia. The Vaccine Adverse Event Reporting System has received 14,072 reports for the HPV-6/11/16/18 vaccine since licensure, with only 7% being serious AEs, about half the average reported for licensed vaccines in general.HPV-6/11/16/18 vaccination was associated with more injection-site pain than placebo but similar incidences of systemic and serious AEs and new medical conditions potentially consistent with autoimmune phenomena. Based on review of post-licensure safety information, the benefits of vaccination to prevent the majority of genital tract precancers and cancers continue to far outweigh its risks.

The potential as prophylactic vaccines of L1-based particles from cutaneous genus alpha human papillomavirus (HPV) types has not been assessed so far. However, there is a high medical need for such vaccines since HPV-induced skin warts represent a major burden for children and for immunocompromised adults, such as organ transplant recipients. In this study, we have examined the immunogenicity of capsomeres and virus-like particles (VLPs) from HPV types 2, 27, and 57, the most frequent causative agents of skin warts. Immunization of mice induced immune responses resembling those observed upon vaccination with HPV 16 L1-based antigens. The antibody responses were cross-reactive but type-restricted in their neutralizing capacities. Application of adjuvant led to an enhanced potential to neutralize the respective immunogen type but did not improve cross-neutralization. Vaccination with capsomeres and VLPs from all four analyzed HPV types induced robust IFNγ-associated T-cell activation. Immunization with mixed VLPs from HPV types 2, 27, and 57 triggered an antibody response similar to that after single-type immunization and capable of efficiently neutralizing all three types. Our results imply that vaccination with combinations of VLPs from cutaneous HPV types constitutes a promising strategy to prevent HPV-induced skin lesions.