Local and systemic responses against ricin toxin promoted by toxoid or peptide vaccines alone or in liposomal formulations

The objective of this study was to develop a vaccine which would ultimately protect man from the lethal effects of inhaled ricin toxin. Porton rats have previously been protected from lethal quantities of inhaled ricin by subcutaneous (s.c.) ricin toxoid vaccine, but not without lung damage. This situation might be improved by an alternative vaccine such as the A chain of ricin, already known to protect against inhaled ricin. Another option would be to improve respiratory tract immunity by local vaccination in conjunction with liposomal formulation with a view to enhancing lung secretion of immune IgA. While boosted s.c. doses of ricin toxoid or A chain produced indistin-guishable systemic immune responses 3 weeks later, when delivered by the intratracheal (i.t.) route, the A chain failed to elicit a specific immune response, unlike ricin toxoid. This situation was overcome by liposomal formulations and although ricin toxoid was readily encapsulated in liposomes, A chain was not. However, by simply mixing A chain and liposomes in the same weight ratio determined for liposomal toxoid, systemic immune responses for each formulation were indistinguishable 1 week after boosting. Ricin antibody responses in lung fluid monitored 1, 3, 7 and 14 days after i.t. challenge with ricin were statistically indistinguishable, but the group vaccinated with liposomal toxoid secreted 28.7% IgA compared with 0.9–14.9% for the A chain liposomal group. From this, it might be anticipated that the lungs would be better protected by liposomally-encapsulated ricin toxoid than by the A chain-liposome mixture.     

Liposomally-encapsulated ricin toxoid vaccine delivered intratracheally elicits a good immune response and protects against a lethal pulmonary dose of ricin toxin

A small study was performed to examine whether the instillation of ricin toxoid vaccine into the lungs of Porton rats offered protection from lethal effects of subsequent intratracheal challenge with ricin toxin. Further, the immune response to liposomally-encapsulated vaccine and the protection offered was compared with vaccine either adsorbed to Alhydrogel adjuvant or as a simple aqueous solution. The formaldehyde-treated ricin toxin vaccine (RTV) was administered at two dose levels, 500 and 100 μg kg⁻¹ body weight to groups of rats, on two occasions by intratracheal instillation. Liposomally-encapsulated vaccine (LRTV) produced a higher titre of ricin-specific antibodies than Alhydrogel-vaccine (ARTV) and vaccine solution. When challenged with 3 LD₅₀ of ricin by intratracheal instillation 7 weeks after the second vaccine instillation, all rats in both LRTV dose groups survived with minimal signs of incapacitation. Analysis of antibody secretion by spleen cells, 14 days post challenge, showed that the IgG isotype in the LRTV group was significantly higher than that in the ARTV and RTV groups and also that the proportion of specific IgA in lung fluid was higher in the LRTV group than in the ARTV and RTV groups. The results of this study indicate that effective vaccinations against inhaled ricin could be achieved with liposomally-encapsulated ricin toxoid, via the lung and should be investigated further.     

Novel vaccine formulations against pertussis offer earlier onset of immunity and provide protection in the presence of maternal antibodies

Whooping cough is a respiratory illness most severe in infants and young children. While the introduction of whole-cell (wP) and acellular pertussis (aP) vaccines has greatly reduced the burden of the disease, pertussis remains a problem in neonates and adolescents. New vaccines are needed that can provide early life and long-lasting protection of infants. Vaccination at an early age, however, is problematic due to the interference with maternally derived antibodies (MatAbs) and the bias towards Th2-type responses following vaccination. Here we report the development of a novel vaccine formulation against pertussis that is highly protective in the presence of MatAbs. We co-formulated pertussis toxoid (PTd) and filamentous hemagglutinin (FHA) with cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN), cationic innate defense regulator (IDR) peptide and polyphosphazene (PP) into microparticle and soluble vaccine formulations and tested them in murine and porcine models in the presence and absence of passive immunity. Vaccines composed of the new adjuvant formulations induced an earlier onset of immunity, higher anti-pertussis IgG2a and IgA titers, and a balanced Th1/Th2-type responses when compared to immunization with Quadracel^®, one of the commercially available vaccines for pertussis. Most importantly, the vaccines offered protection against challenge infection in the presence of passively transferred MatAbs.     

Enhanced immune protection by a liposome-encapsulated recombinant respiratory syncytial virus (RSV) vaccine using immunogenic lipids from Deinococcus radiodurans

The radiation-resistant bacterium, Deinococcus radiodurans contains a variety of phospho-, glyco- and phosphoglycolipids, the structures of which appear to be largely unique in nature. We show here that such lipids are immunogenic when administered as liposomes intranasally in mice, as evidenced by the induction of serum antibodies which recognize D. radiodurans lipids but not other lipids by thin layer chromatographic immunostaining. By modifying a liposomal vaccine against respiratory syncytial virus (RSV) we find that vaccine efficacy is markedly enhanced by the inclusion of lipids isolated from D. radiodurans. Using dioleoylphosphatidylcholine (DOPC) or D. radiodurans lipids, liposomes were prepared which encapsulated a soluble fragment of the RSV G protein (G(128-188)) fused with a portion of the bacterial thioredoxin (Trx) protein. Mice immunized intranasally with D. radiodurans liposomes showed markedly greater protection against RSV challenge over mice immunized with DOPC liposomes. Enhanced vaccine efficacy was achieved using liposomes prepared from either whole D. radiodurans lipids or from a single isolated phosphoglycolipid previously identified as alpha-galactosylphosphatidylglyceroylalkylamine (lipid 7). Mice immunized and protected against RSV challenge were free of pulmonary eosinophilic infiltration, an undesirable consequence of many RSV vaccines. The results provide further support for liposome-based vaccines for RSV and underline the importance of lipid composition in liposome formulations.     

Evidence for enhanced central memory priming by live Mycobacterium bovis BCG vaccine in comparison with killed BCG formulations

Development of cattle vaccines against bovine tuberculosis is a GB research priority. Recently, it has been shown that formalin-killed Bacille Calmette-Guérin (BCG) delivered with the liposomal adjuvant NAX687 imparted significant protection against Mycobacterium bovis infection in the guinea pig aerosol infection model. Extending these studies, we inoculated calves with live BCG, formalin-killed BCG and formalin-killed BCG formulated in NAX687. Live and killed BCG vaccine formulations induced primary effector T-cell populations comparably, both killed BCG formulations also induced potent humoral immune responses. In contrast, live BCG generated enhanced central memory responses against the protective antigen Ag85A whilst killed BCG-induced such responses only poorly. However, the poor capacity of killed BCG to generate central memory could be partially overcome by formulation with NAX687. Measurement of central memory responses induced by TB vaccine candidates in cattle may provide a useful correlate of protection and warrants further investigation in challenge experiments.     

Laboratory investigation of immune responses to acellular pertussis vaccines when used for boosting adolescents after primary immunisation with whole cell pertussis vaccines: a comparison with data from clinical study

The lack of unequivocal immunological correlates of human protection and an absence of a validated animal model for acellular pertussis vaccines, compounded by limited opportunity to undertake efficacy studies in humans and laboratory evaluation side by side, has made it difficult to compare vaccines and formulations. In the present study, the effect on the booster response to pertussis in adolescents primed in infancy with whole cell pertussis vaccine, of three low dose acellular pertussis/diphtheria/tetanus toxoid (TdPa) formulations with or without inactivated poliomyelitis vaccine (IPV) components, was investigated. To assess the relationship between laboratory vaccine evaluation and clinical trial performance, parallel evaluation of the same TdPa vaccines were carried out in a mouse booster model with whole cell pertussis vaccine priming. Prior to boosting, the clinical subjects had low cell mediated immune responses (CMI) responses to pertussis vaccine components. After boosting, all TdPa formulations stimulated CMI responses to the pertussis vaccine components assessed. The booster responses to the pertussis antigens remained skewed towards Th1 type even though acellular pertussis vaccines were used. In general the antibody and CMI responses to pertussis antigens in the mouse model followed the trend seen in the human subjects. Protection against aerosol challenge with virulent Bordetella pertussis was related to the magnitude of the antibody and CMI responses in the mouse model. As in the human subjects, the responses remained skewed towards Th1 type.     

RiVax, a recombinant ricin subunit vaccine, protects mice against ricin delivered by gavage or aerosol

Ricin is a plant toxin that is a CDC level B biothreat. Our recombinant ricin A chain vaccine (RiVax), which contains mutations in both known toxic sites, has no residual toxicity at doses at least 800 times the immunogenic dose. RiVax without adjuvant given intramuscularly (i.m.) protected mice against intraperitoneally administered ricin. Furthermore the vaccine without alum was safe and immunogenic in human volunteers. Here we describe the development of gavage and aerosol ricin challenge models in mice and demonstrate that i.m. vaccination protects mice against ricin delivered by either route. Also RiVax protects against aerosol-induced lung damage as determined by histology and lung function tests.     

Efficacy of an ovine caseous lymphadenitis vaccine formulated using a genetically inactive form of the Corynebacterium pseudotuberculosis phospholipase D

Caseous lymphadenitis (CLA) is an economically significant disease of sheep caused by the gram-positive bacterium Corynebacterium pseudotuberculosis. CLA vaccines are currently formulated using formalin inactivated culture supernatants that are rich in the C. pseudotuberculosis phospholipase D (PLD) exotoxin. One alternative to chemical detoxification is to inactivate the PLD genetically. This procedure not only provides a means to remove an onerous chemical treatment step but also the opportunity to increase gene expression, therefore improve protein yields. Using site-specific mutagenesis the C. pseudotuberculosis PLD was inactivated by substituting a serine residue at histidine 20 within the enzyme active site. CLA vaccine formulated using genetically inactivated PLD protected 44% of sheep against C. pseudotuberculosis challenge compared with 95% protection offered by the formalin inactivated preparation. Since there was no apparent difference in immune response mounted by vaccinated sheep the reason for this variation in vaccine efficacy remains unclear. Although genetic inactivation can be a convenient means to produce toxoid vaccines its use to develop a new CLA vaccine provided no net benefit over the conventional formulation.     

Protection against pneumonic plague following oral immunization with a non-replicating vaccine

Yersinia pestis is a dangerous bacterial pathogen that when inhaled can rapidly induce fatal pneumonic plague. Thus, there is a need for stable, safe, and easily administered mucosal vaccines capable of eliciting effective protection against pulmonary Y. pestis infections. Cationic liposome–nucleic acid complexes (CLDC) have been shown previously to be effective vaccine adjuvants for parenteral immunization, but have not been previously evaluated for use in oral immunization. Therefore, we investigated the ability of an orally administered CLDC adjuvanted vaccine to elicit protective immunity against lethal pneumonic plague. C57Bl/6 mice were vaccinated orally or subcutaneously using 10 μg Y. pestis F1 antigen combined with CLDC and immune responses and protection from challenge was assessed. We found that oral immunization elicited high titers of anti-F1 antibodies, equivalent to those generated by parenteral immunization. Importantly, orally immunized mice were protected from lethal pulmonary challenge with virulent Y. pestis for up to 18 weeks following vaccination. Vaccine-induced protection following oral immunization was found to be dependent primarily on CD4+ T cells, with a partial contribution from CD8+ T cells. Thus, CLDC adjuvanted vaccines represent a new type of orally administered, non-replicating vaccine capable of generating effective protection against pulmonary infection with virulent Y. pestis.     

Lipophilic quaternary ammonium salt acts as a mucosal adjuvant when co-administered by the nasal route with vaccine antigens.

Nasal administration of vaccines is an attractive approach which offers several significant advantages over traditional intramuscular vaccine delivery. These advantages include easier administration and induction of immune responses in the mucosal secretions of the body. In this study we describe a new potent nasal adjuvant, dimethyldioctadecylammonium bromide (DDA), that induces both mucosal and systemic immune responses when co-administered with diphtheria toxoid (DT), tetanus toxoid (TT) and BBG2Na antigens. In particular, we show that the nasal delivery of recombinant fragment (BBG2Na) of the G protein of respiratory syncytial virus (RSV) mixed with DDA induces both local and systemic anti-RSV immune responses and protects against viral challenge. Furthermore, we provide evidence that the DDA+BBG2Na vaccine does not induce lung immunopathology upon subsequent RSV challenge.     

Protective immunity induced by an inhaled SARS-CoV-2 subunit vaccine

Targeting the site of infection is a promising strategy for improving vaccine effectivity. To date, licensed COVID-19 vaccines have been administered intramuscularly despite the fact that SARS-CoV-2 is a respiratory virus. Here, we aim to induce local protective mucosal immune responses with an inhaled subunit vaccine candidate, ISR52, based on the SARS-CoV-2 Spike S1 protein. When tested in a lethal challenge hACE2 transgenic SARS-CoV-2 mouse model, intranasal and intratracheal administration of ISR52 provided superior protection against severe infection, compared to the subcutaneous injection of the vaccine. Interestingly for a protein-based vaccine, inhaled ISR52 elicited both CD4 and CD8 T-cell Spike-specific responses that were maintained for at least 6 months in wild-type mice. Induced IgG and IgA responses cross-reacting with several SARS- CoV-2 variants of concern were detected in the lung and in serum and protected animals displayed neutralizing antibodies. Based on our results, we are developing ISR52 as a dry powder formulation for inhalation, that does not require cold-chain distribution or the use of needle administration, for evaluation in a Phase I/II clinical trial.     

Head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model

In order to study the influence of antigen composition, spatial organization of antigen and the route of administration, four cell culture-derived, inactivated, nonadjuvanted influenza vaccine formulations, i.e. whole inactivated virus (WIV), split, subunit and virosome vaccines were prepared from a single antigen batch. We directly compared the immunogenicity and efficacy of these vaccine formulations after intramuscular (i.m.) or intranasal (i.n.) administration in mice. Prime and boost vaccination were followed by a potentially lethal homologous aerosol challenge. For all vaccines, the i.m. route induced higher serum humoral immune responses as compared to the i.n. route and protected all mice against challenge at a dose of 5 microg. Upon i.n. immunization only WIV and split vaccines induced detectable IgG titers and partial protection against challenge but only very low HI titers were induced in almost all mice. WIV induced mainly IgG2a/c titers via both routes, whereas split vaccine induced exclusively IgG1 titers via both routes. Subunit and virosome vaccines induced exclusively IgG1 via the i.m. route. Mucosal sIgA levels were only detected after i.n. vaccination with WIV. Furthermore, vaccines containing all viral components (WIV and split vaccine) induced higher serum HI titers and serum antibody titers than subunit and virosome vaccines. The differences in magnitude and quality of immune responses of split and WIV, having the same composition, are likely related to their distinct spatial organization. In conclusion, the direct comparison between WIV, split, subunit and virosomes, shows that the differences in immune responses between these well known influenza vaccines can be explained by both the composition and particulate structure of these vaccine formulations.     

Single dose intranasal immunization with ISCOMATRIX™ vaccines to elicit antibody-mediated clearance of influenza virus requires delivery to the lower respiratory tract

The effectiveness of single dose, intranasally delivered vaccines comprising detergent-disrupted inactivated influenza virus (split virus) and ISCOMATRIX™ adjuvant was examined in mice. Vaccines formulated with adjuvant required 10- to 100-fold less split virus antigen to induce pulmonary protection following viral challenge when compared to vaccines containing split virus alone. Furthermore, those formulated with ISCOMATRIX™ adjuvant elicited specific antibody in serum, saliva, vaginal, nasal and lung fluids when delivered to the entire respiratory tract. No specific antibody was detected in serum or mucosal samples, however, when the same vaccines were delivered using a procedure that restricted the inoculum to the nasal passages. Good protective responses can thus be achieved with only a single intranasal inoculation of influenza vaccine formulated with adjuvant, providing the vaccine can access sites of immune induction in the lower respiratory tract.     

Immunogenicity of IRIV- versus alum-adjuvanted diphtheria and tetanus toxoid vaccines in influenza primed mice

The immunogenicity and protectivity of two different toxoid vaccines were compared in mice. In one formulation, toxoids (diphtheria or tetanus) were adsorbed to alumoxid, whereas in the other formulation the toxoids were crosslinked to immunopotentiating reconstituted influenza virosomes (IRIVs). A preimmunization with influenza antigens is necessary for a good anti-toxoid antibody response when the IRIV formulation was administered. After two immunizations with the IRIV- or alum-based vaccines, the IRIV-based formulation induced a higher humoral immune response than toxoids adsorbed to alum. Using an in vitro test, diphtheria toxin neutralizing antibodies were tested. Di-IRIV induced a significantly (p = 0.002) higher titer of diphtheria toxin neutralizing antibodies than Di-alum. Tetanus challenge experiments showed, that the IRIV-based tetanus vaccine induced a threefold higher titer of protective antibodies than the tetanus toxoid adsorbed to alum. Therefore, the IRIV-based formulations appeared to be superior to the alum-based vaccines in terms of immunogenicity and protectivity.     

Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge

Mucosal (oral) immunization of mice with carrier-delivered ricin toxoid (RT) vaccine was accomplished by one long (7 weeks) or two short (4 weeks) immunization schedules. For the long and short immunization schedule two lots of vaccine were administered prepared with the same procedure but at different occasions. The long schedule consisted of a total of seven doses of 50 microg of vaccine in microencapsulated (lot #108) or aqueous form administered on days 1, 2, 3, 28, 29, 30 and 49. With the short schedule a total of seven or six doses of 25 microg (lot #111) were administered on days 1, 2, 3, 14, 15, 16 and 30, or on 1, 2, 14, 15, 30, 31 and 32, respectively. Mice immunized orally with the long schedule, 50 microg of RT vaccine incorporated into poly-DL-lactide-co-glycolyde (DL-PLG) microspheres (MS) produced serum IgG, IgG2a and IgA ELISA antibodies. All mice immunized with RT in DL-PLG MS (RT-MS) were protected against a lethal ricin aerosol challenge. In contrast, with the same schedule and with the same dose, the aqueous vaccine (RT) failed to stimulate IgG, IgG2a and IgA antibodies, and these mice were not protected against an aerosol ricin toxin challenge. With the shorter immunization scheme, seven doses of 25 microg RT-MS stimulated a significant, though reduced, protection with the microencapsulated, but not with the aqueous vaccine. When the first and second 3-day cycles of the short immunization schedule was reduced to two doses, and the 3-day cycle was administered at the end of the schedule, neither RT-MS nor RT stimulated protection against the challenge. These results indicated that successful oral immunization with RT-MS depended on both the dose and the schedule, consisting of three consecutive days of administration in two cycles, 4 weeks apart. Altering this schedule and the dose, resulted in a reduced protection or no protection at all. Furthermore, under the conditions of this study, the advantage of the microencapsulated RT vaccine over the aqueous vaccine for effective oral immunization was well demonstrated.     

Intradermal administration of RiVax protects mice from mucosal and systemic ricin intoxication

Ricin toxin is a CDC level B biothreat. We have developed a ricin vaccine, RiVax, which is a recombinant mutant of ricin A chain. RiVax is safe, immunogenic and protective in mice when administered intramuscularly (IM). We have now attempted to increase the utility and immunogenicity of RiVax by administering it intradermally (ID) with or without alum. Without alum, Rivax administered by the ID and IM routes was equally immunogenic and protective. With alum, ID vaccinations were more immunogenic and protective against both systemic and mucosal challenge with ricin and superior in protecting animals from ricin-induced lung damage.     

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.     

Saponins from the Spanish saffron Crocus sativus are efficient adjuvants for protein-based vaccines

Protein and peptide-based vaccines provide rigorously formulated antigens. However, these purified products are only weakly immunogenic by themselves and therefore require the addition of immunostimulatory components or adjuvants in the vaccine formulation. Various compounds derived from pathogens, minerals or plants, possess pro-inflammatory properties which allow them to act as adjuvants and contribute to the induction of an effective immune response. The results presented here demonstrate the adjuvant properties of novel saponins derived from the Spanish saffron Crocus sativus. In vivo immunization studies and tumor protection experiments unambiguously establish the value of saffron saponins as candidate adjuvants. These saponins were indeed able to increase both humoral and cellular immune responses to protein-based vaccines, ultimately providing a significant degree of protection against tumor challenge when administered in combination with a tumor antigen. This preclinical study provides an in depth immunological characterization of a new saponin as a vaccine adjuvant, and encourages its further development for use in vaccine formulations.     

Vaccine-induced intestinal immunity to ricin toxin in the absence of secretory IgA

The RNA N-glycosidase ribosome inactivating proteins (RIPs) constitute a ubiquitous family of plant- and bacterium-derived toxins that includes the category B select agents ricin, abrin and shiga toxin. While these toxins are potent inducers of intestinal epithelial cell death and inflammation, very little is known about the mechanisms underlying mucosal immunity to these toxins. In the present study, we report that secretory IgA (SIgA) antibodies are not required for intestinal immunity to ricin, as evidenced by the fact that mice devoid of SIgA, due to a mutation in the polymeric immunoglobulin receptor, were impervious to the effects of intragastric toxin challenge following ricin toxoid immunization. Furthermore, parenteral administration of ricin-specific monoclonal IgGs, directed against either ricin's enzymatic subunit (RTA) or ricin's binding subunit (RTB), to wild type mice was as effective as monoclonal IgAs with comparable specificities in imparting intestinal immunity to ricin. These data are consistent with reports from others demonstrating that immunization of mice by routes known not to induce mucosal antibody responses (e.g., intramuscular and intradermal) is sufficient to elicit protection against both systemic and mucosal ricin challenges.     

Comparison of different doses of antigen for intradermal administration in pigs: the Actinobacillus pleuropneumoniae model

The intensity of antibody responses and level of protection against challenge infection induced by Actinobacillus pleuropneumoniae (APP) were compared in piglets vaccinated intramuscularly with different vaccination doses of a subunit vaccine. Secondary antibody responses to APP exotoxins induced by the intradermally administered diluted vaccines did not differ from those induced by undiluted vaccines administered either intradermally or intramuscularly. The level of protection measured by the clinical course of challenge infection to the extent of lung lesions was significantly higher in animals vaccinated with a three-time diluted vaccine in comparison with animals that were administered intramuscularly or intradermally with the most concentrated or the most diluted vaccines.