Serum and mucosal immune responses to an inactivated influenza virus vaccine induced by epidermal powder immunization

Both circulating and mucosal antibodies are considered important for protection against infection by influenza virus in humans and animals. However, current inactivated vaccines administered by intramuscular injection using a syringe and needle elicit primarily circulating antibodies. In this study, we report that epidermal powder immunization (EPI) via a unique powder delivery system elicits both serum and mucosal antibodies to an inactivated influenza virus vaccine. Serum antibody responses to influenza vaccine following EPI were enhanced by codelivery of cholera toxin (CT), a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs (CpG DNA), or the combination of these two adjuvants. In addition, secretory immunoglobulin A (sIgA) antibodies were detected in the saliva and mucosal lavages of the small intestine, trachea, and vaginal tract, although the titers were much lower than the IgG titers. The local origin of the sIgA antibodies was further shown by measuring antibodies released from cultured tracheal and small intestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays. EPI with a single dose of influenza vaccine containing CT or CT and CpG DNA conferred complete protection against lethal challenges with an influenza virus isolated 30 years ago, whereas a prime and boost immunizations were required for protection in the absence of an adjuvant. The ability to elicit augmented circulating antibody and mucosal antibody responses makes EPI a promising alternative to needle injection for administering vaccines against influenza and other diseases.     

Immune responses of mice to influenza subunit vaccine in combination with CIA07 as an adjuvant

CIA07 is an immunostimulatory agent composed of E. coli DNA fragments and modified LPS lacking the lipid A moiety. In this study, we investigated whether CIA07 promotes immune responses as an adjuvant to the influenza subunit vaccine. Balb/c mice were immunized intramuscularly once or twice at a 4-week interval with the trivalent influenza subunit vaccine antigen alone or in combination with CIA07 as adjuvant. Antigen-specific serum antibody titers and hemagglutination-inhibition (HI) antibody titers were assessed. At 4 weeks after each immunization, the antigen-specific total serum IgG antibody titer in mice receiving CIA07 was 2 to 3 times higher than that in animals administered antigen alone (P<0.05). The CIA07-treated group additionally displayed higher HI antibody titers against each of the 3 vaccine strains, compared to the antigen group. Animals receiving antigen alone displayed barely detectable antigen-specific serum IgG2a antibody titers. In contrast, coadministration of CIA07 with antigen led to significantly enhanced IgG2a antibody responses, suggesting that CIA07 stimulates a Th1-type immune response. Moreover, the CIA07-treated group displayed a marked increase in the number of interferon gamma-producing CD8(+) T cells in splenocytes. These data collectively demonstrate that CIA07 has the ability to induce both Th1-type cellular and Th2-type humoral immune responses to the influenza subunit vaccine, and support its potential as an effective adjuvant to the influenza vaccine.     

Measurement of IgM responses to a subunit influenza A vaccine by sucrose-gradient centrifugation and membrane-filtration enzyme immunoassays

IgM responses to a deoxycholate-split influenza vaccine containing the surface antigens of the H3N2 virus A/Philippines/2/82 were studied in five volunteers, three of whom were seronegative by haemagglutination inhibition (HI) tests. Responses were measured by a sucrose-gradient centrifugation technique, in which IgM-specific HI activity was computed as a proportion of total IgM and IgG-specific HI activity, and by a membrane filtration-enzyme immunoassay (MF-EIA). Responses could be detected in all volunteers when measured by sucrose-gradient centrifugation within 1-2 weeks, and the IgM induced was 5-40% of total HI-specific activity. The response was also observed in the presence of low levels of pre-existing antibody. Levels of IgM antibody, when measured by MF-EIA, could be easily detected in two of the volunteers, while those of two others were very low and there was no response in a fifth. No biphasic virus-specific response to vaccination, involving first IgM and then IgG, could be measured by either technique. From these studies, the sucrose-gradient fractionation technique appears to be the more sensitive procedure.     

Towards a subunit influenza vaccine prepared by affinity chromatography on immobilized lectin

Disintegration of influenza virions with 0.2% w/v sodium deoxycholate releases haemagglutinin and neuraminidase, which in the presence of detergent are both adsorbed to the lectin fromCrotalaria juncea, coupled to Sepharose 2B.The binding is mediated through galactosyl residues on haemagglutinin and neural-minidase, which are completely displaced in 0.2 M lactose and co-elute in a narrow zone.The immunogenicity of haemagglutinin and neuraminidase in mice is markedly increased after adsorption onto lipid, particles constituting “intralipid” (Kabi Vitrum, Stocholm, Sweden).     

Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.     

Reaction of the biologically active points of the skin to immunization with typhus vaccine

Electroacupuncture diagnosis was used to demonstrate the reaction of biologically active points of the skin (BAPS) to immunization with typhus vaccine. In 7 persons immunized with chemical and live (strain E) typhus vaccines, the reaction differed significantly from the initial parameters and other measurement data obtained in a group of unvaccinated subjects. The reaction of the BAPS was noticeable starting from the first day after vaccination and was far ahead of seroimmune shifts detectable in the host. The best correlation was established between the seroimmune response and the activity of the acupuncture points on meridians X and I (a positive correlation) and on meridians III, VIII and XI (a negative correlation).     

Adsorbed influenza chemical vaccine in the immunization of schoolchildren

When used for the immunization of healthy children aged 7-14 year, adsorbed chemical influenza vaccine produced at the Mechnikov Research Institute for Vaccines and Sera in Ufa has proved to be safe, faintly reactogenic and antigenically very potent. The optimum doses for immunizing children aged 7-10 and 11-14 years have been selected.     

Enhancement of DTH response by cholera toxin B subunit inoculated intranasally together with influenza HA vaccine

The effects of B subunit of cholera toxin (CTB) on delayed-type hypersensitivity (DTH) response to influenza vaccine derived from influenza virus A/PR/8/34 (PR-8, HlNl) virus were investigated in B10 mice that were immunized intranasally with both influenza vaccine and CTB. The result showed that intranasal inoculation of this combination augmented DTH response to influenza vaccine, which reached its peak 6 days after inoculation, and also induced accelerated DTH response upon a second inoculation of influenza vaccine alone 4 weeks later, that the cross-reactive DTH response to PR-8 vaccine was elicited by the injection of the different influenza A-type virus vaccine into the footpad of the vaccinated mice, but was not by influenza B-type virus vaccine, that the DTH-mediating T cells were detected selectively in the lungs of mice that received the nasal inoculation of the vaccine and CTB together, but that subcutaneous inoculation of this combination failed to induce DTH-mediating T cells in the lungs. These results, together with the previous papers (Tamura et al, Vaccine 7: 257-262; 314-320, 1989), suggest that CTB could augment both humoral and DTH responses against influenza vaccine in the respiratory mucosal tract.     

Interrelationships between serum and secretory antibodies in immunization with live influenza vaccine]

A study was made of the accumulation of antibodies in the blood serum and the secretions of the respiratory tracts of persons immunized with the living influenza vaccine. The duration of inductive phase and the dynamics of the antibody accumulation in the secretions occurred irrespective of their initial level in the blood sera, this pointing to the autonomic character of the local immunity system. On the other hand the functional condition of the system of local immunity influenced the intensity of the antibody formation in the system of the general immunity. If before the immunization the antibody titre in the secretions were 1:4 and greater, the antibody accumulation in the blood sera took place less intensively. An analogous phenomenon was also observed when the antibodies were absent in the secretions before the immunization, but their formation took place as soon as the first week after it. The mechanism of this peculiar "competition for the antigen" of the systems of local and general immunity consisted in the neutralization of the influenza virus in the area of the porta of infection.     

Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza

Background

The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored.

Methods and Results

A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs.

Conclusions

The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.     

Validation of immunity induced by inactivated CCPP vaccine with different adjuvants

The study was conducted in the premises National Veterinary Institute (NVI) to validate the immunity induced by inactivated F38 antigen adjuvated with saponin and Montanide ISA 50 and combined with and without anthrax vaccine.Post-inoculation reactions; pyrogenic effects, safety and inocuity of the vaccines were assessed. Increased body temperature and local edematous reactions were seen in animals inoculated with saponin adjuvated CCPP vaccine (100%) while 20% of the goats in ISA 50 adjuvated group showed local reaction. Sera collected from day 0 to 10th week were tested to assess the sero-conversion using monoclonal antibody based B-ELISA technique. Saponin adjuvated groups, in both monovalent CCPP and in the combined CCPP with anthrax vaccine showed a higher mean percentage of inhibition value as compared with ISA 50 adjuvated vaccine.After 8 months of post vaccination, contact challenge trial was conducted in 66 experimentally vaccinated and 20 negative control goats combined with 15 actively CCPP sick goats. Various clinical signs were recorded daily, autopsy was done on died goats and the live goats were sacrificed after 2 months of contact. The side by side samples from thoracic exudates, lung and mediastinal and bronchial lymph nodes were collected from goats shown to have developed indicative CCPP lesion for isolation and F38 antigen detection.The present experimental study indicated that application of inactivated and adjuvated CCPP vaccine significantly reduced the morbidity and development of lesions (P < 0.001). Among vaccinated groups CCPP + anthrax + saponin showed better protection, with low rate of nasal discharge and cough at 33% and 28.6%, respectively, and protection level of 94.1% from death and 65% from lung lesion development. However, the variation in protection among the vaccinated groups was not significant (P > 0.05).These findings disclosed that inactivated CCPP vaccine adjuvated with saponin and ISA 50 significantly reduce morbidity and mortality of goats due to CCPP and also indicated the importance of utilization of ISA 50 as alternative adjuvant to minimize post-vaccinal reactions encountered in use of saponin as adjuvant.     

流感裂解疫苗H3N2-抗H3N2免疫原性复合物滴鼻诱导小鼠黏膜免疫应答的研究

目的研究流感裂解病毒疫苗抗原抗体复合物滴鼻诱生小鼠黏膜免疫应答.方法分别以15μg H3N2、H3N2-CpG、H3N2-鼠抗H3N2及H3N2-PEG滴鼻免疫小鼠,检测肺泡灌洗液抗H3N2 IgA、血清抗H3N2 IgG效价.取免疫小鼠脾细胞,体外抗原刺激,用定量酶联免疫吸附试验(ELISA)检测上清液IFN-γ及IL-4分泌水平.结果H3N2-抗H3N2免疫原性复合物诱生的抗H3N2 IgA效价明显高于H3N2单独免疫组(P＜0.01),而与H3N2-CpG组无显著性差异.此外,复合物诱生的血清抗H3N2也高于H3N2单独免疫组(P＜0.05).H3N2-CpG组诱生的IFN-γ水平明显升高,而其他组之间无明显差异.结论流感病毒血凝素抗原抗体复合物、血凝素抗原加CpG佐剂可以诱生较强的局部黏膜免疫和体液免疫.这两组诱生的IgA效价均明显高于H3N2单独免疫组.另外,H3N2-CpG组小鼠的脾脏细胞经特异性抗原诱导后培养上清液中的IFN-γ水平明显升高.     

Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens

Intranasal immunization with inactivated influenza virus vaccine can provide protective immunity, whereas many other antigens are less effective when used for mucosal immunization. To determine whether influenza virus could enhance immune responses to an antigen coadministered to a mucosal surface, we studied the intranasal immunization of mice with a mixture of simian-human immunodeficiency virus (SHIV) virus-like particles (VLPs) and inactivated influenza virus. Compared to mice immunized with SHIV VLPs alone, mice coimmunized with SHIV VLPs and inactivated influenza virus showed significant increases in serum immunoglobulin G (IgG) and mucosal IgA antibodies specific to the human immunodeficiency virus envelope protein, neutralizing activities, numbers of gamma interferon- and interleukin 4-secreting lymphocytes, and cytotoxic-T-lymphocyte activities. The levels of enhancement of immune response by coimmunization with inactivated influenza virus were equivalent to those induced by inclusion of immunostimulatory CpG oligodeoxynucleotides (CpG DNA). We also observed that SHIV VLPs bind to influenza virus virions, forming mixed aggregates. These results indicate that inactivated influenza virus can play a role as a mucosal adjuvant to coadministered antigens.     

Modulation of antigen-specific humoral responses in rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants

An important limitation of DNA immunization in nonhuman primates is the difficulty in generating high levels of antigen-specific antibody responses; strategies to enhance the level of immune responses to DNA immunization may be important in the further development of this vaccine strategy for humans. We approached this issue by testing the ability of molecular adjuvants to enhance the levels of immune responses generated by multicomponent DNA vaccines in rhesus macaques. Rhesus macaques were coimmunized intramuscularly with expression plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs encoding human immunodeficiency virus Env and Rev and simian immunodeficiency virus Gag and Pol proteins. We observed that the cytokine gene adjuvants (especially IL-2 and IL-4) significantly enhanced antigen-specific humoral immune responses in the rhesus macaque model. These results support the assumption that antigen-specific responses can be engineered to a higher and presumably more desirable level in rhesus macaques by genetic adjuvants.     

Vault nanocapsules as adjuvants favor cell-mediated over antibody-mediated immune responses following immunization of mice

Background

Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier.

Methodology and Principal Findings

We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8⁺ and CD4⁺ memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8⁺ memory T cells and production of IFNγ plus CD4⁺ memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes.

Conclusions/Significance

These experiments show that vault nanocapsules induced strong anti-OVA CD8⁺ and CD4⁺ T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8⁺ and CD4⁺ T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.     

MF59 and Pam3CSK4 boost adaptive responses to influenza subunit vaccine through an IFN type I-independent mechanism of action

The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.