Comparison of the quality of protection elicited by toxoid and peptide liposomal vaccine formulations against ricin as assessed by markers of inflammation.

Ricin is a very toxic substance which inhibits protein synthesis and produces severe tissue damage and inflammation. It is very potent when inhaled as an aerosol and protection has been examined in a series of studies using vaccine candidates including a formaldehyde inactivated ricin toxoid and the A chain of ricin, a polypeptide equivalent to half of the toxin molecule. Initially, subcutaneous injections of both compounds were found to protect against inhaled ricin but not without some subsequent adverse signs. Intra-pulmonary vaccination using liposomal formulations of these compounds was investigated with a view to improving lung condition following challenge. Using the humoral and local pulmonary immune responses as indices of vaccine performance, no significant difference between toxoid or peptide vaccines was found. In the third and current study, the quality of lung protection by vaccines was assessed using markers of inflammation. Thus, the profiles of inflammatory cell and protein influx into the lung were determined following intratracheal (i.t.) challenge with ricin of rats treated with liposomal vaccine formulations. Results showed that liposomal ricin toxoid offered a better quality of protection with a significantly lower influx of polymorphonuclear leucocytes (neutrophils) and little pulmonary oedema compared with the A chain/liposome formulation. Further, there was no significant difference between the quality of protection offered by the A chain when administered subcutaneously or locally into the lung by i.t. instillation. Liposomal ricin toxoid is a good candidate vaccine and optimised pulmonary delivery by inhalation should be further examined.     

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.     

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.     

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.     

The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant

Systemic and mucosal immune responses were effectively induced following the subcutaneous administration of Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine in a formulation containing the active form of vitamin D3. IgA and IgG antibodies with specificity for both the protein and oligosaccharide components of the vaccine were detectable in mucosal secretions following immunization. The IgA and IgG mucosal antibodies were produced locally, and were functional as demonstrated by their diphtheria toxin neutralizing activity. Our data suggests that subcutaneous tissues can effectively serve as effective antigen presenting sites for both mucosal and systemic immune responses to antigens administered in combination with vitamin D3.     

Immunization of colorectal cancer patients with recombinant baculovirus-derived KSA (Ep-CAM) formulated with monophosphoryl lipid A in liposomal emulsion, with and without granulocyte-macrophage colony-stimulating factor

KSA (Ep-CAM) is highly expressed by colorectal cancers. The safety and immunologic effects of a vaccine consisting of recombinant baculovirus-derived KSA formulated with monophosphoryl lipid A (MPL) in liposomes and emulsified in mineral oil were evaluated, with and without co-administration of granulocyte-macrophage colony-stimulating factor (GM-CSF). Eleven patients with metastatic colorectal cancer received three subcutaneous (s.c.) injections of the vaccine at 4-week intervals. Six patients were randomized to also receive human recombinant GM-CSF (rGM-CSF) by subcutaneous injection daily for 4 days with each vaccination. Immunizations with and without rGM-CSF were well tolerated. Seven of the 11 patients developed significant KSA-specific cellular immune responses as assessed by lymphoproliferation and interferon-gamma (IFN-gamma) ELISPOT assays. All nine tested patients developed positive delayed type hypersensitivity reactions. Eight of the 11 patients developed KSA-specific antibody responses. The highest levels of cellular immune responses were observed in patients who received GM-CSF. Immunization with baculovirus-derived KSA formulated with monophosphoryl lipid A in liposomal emulsion is safe and can elicit KSA-specific immune responses. Co-administration of GM-CSF with this formulation is an effective method of generating KSA-specific T-helper (Th) 1-associated cellular immune responses.     

ProJuvant (Pluronic F127/chitosan) enhances the immune response to intranasally administered tetanus toxoid.

The potential to generate both a systemic and local immune response makes the mucosal system an attractive site for immunization. However, mucosal administration of protein and peptide antigens generally results in a poor immune response. Successful mucosal vaccination is therefore largely dependent on the development of effective mucosal adjuvants. In this study we have examined the effect of mucosal administration of tetanus toxoid (TT) in the presence of a non-ionic block copolymer, Pluronic F127 (F127), with chitosan or lysophosphatidylcholine (LPC) on the systemic and mucosal immune response. Balb/c mice, immunized intraperitoneally (i.p.) with TT and boosted intranasally (i.n.) with TT in F127/chitosan, demonstrated a significant enhancement in the systemic anti-TT antibody response compared to mice boosted i.n. with TT in PBS or mice boosted i.n. with TT in F127/LPC. We determined the antigen specific IgA response in the nasal and lung washes of these animals and found a significant increase in anti-TT mucosal IgA response in the group boosted with TT in F127/chitosan. Similarly, mice immunized and boosted i.n. with TT in F127/chitosan had a significant enhancement of their systemic anti-TT IgG and mucosal IgA antibody responses compared to the animals immunized and boosted i.n. with TT in PBS or TT in F127/LPC. The results of these studies suggest that F127/chitosan represents a novel mucosal vaccine delivery system, consisting of two components, that appear to exert an additive or synergistic effect on the immune response.     

Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses

The induction of mucosal immune responses by a liposome-formulated Y. pestis vaccine (formaldehyde-killed whole cell vaccine; KWC) was evaluated. We demonstrated that intranasal immunization of mice with Y. pestis KWC vaccine, formulated with liposomes, significantly enhanced mucosal immune responses in the lung when compared to the responses induced with KWC vaccine alone. These immune responses were characterized by increased titres of specific IgA and IgG in mucosal secretions (lung and nasal washes), and an increased frequency of specific antibody-secreting cells in the lungs. In addition, antigen-specific proliferative responses and IFN-gamma-secreting cells were also significantly enhanced in the spleens of mice immunized with the KWC vaccine formulated in liposomes. Animals that were immunized intranasally with the KWC vaccine showed significant protection against an intranasal challenge with Y. pestis. These results highlight the importance of mucosal administration of vaccine antigens to stimulate immunity in the respiratory tract and demonstrate that liposome formulations can improve the effectiveness of conventional vaccines.     

Influenza immunization: intranasal live vaccinia recombinant contrasted with parenteral inactivated vaccine.

To compare the efficacy and duration of the immune response to local and systemic vaccination, Balb/c mice were vaccinated either intraperitoneally (i.p.) with an inactivated A/PR/8/34 (H1N1) vaccine or intranasally (i.n.) with a vaccinia recombinant containing the H1 gene of influenza. The i.p. inactivated vaccine stimulated high serum IgG anti-influenza titres and protected the lungs against viral challenge for the duration of the experiment (17 months). Little nasal wash IgA was induced and the noses were susceptible to challenge. Animals vaccinated i.n. with the recombinant had lower serum IgG titres and the lungs showed poor protection against challenge. Nasal wash IgA titres were higher, however, and the noses were largely protected from viral challenge for 17 months.     

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.     

A lyophilized formulation of RiVax,a recombinant ricin subunit vaccine,retains immunogenicity

Ricin is a CDC level B biothreat. Our recombinant ricin A chain vaccine (RiVax) contains two mutations, rendering it non-toxic at high doses. Frozen or alum formulations of RiVax protected mice against ricin administered by injection, gavage or aerosol. Without alum, RiVax was safe and immunogenic in rabbits and human volunteers. For military use, the predominant target group, it would be optimal not to require a cold chain for transport and storage. We have now developed a lyophilized formulation and demonstrated stability and efficacy for at least 1 year stored refrigerated or at room temperature administered with or without alum.     

Liposome fusogenicity and entrapment efficiency of antigen determine the Th1/Th2 bias of antigen-specific immune response

Liposomes, either alone or in combination with additional immunostimulatory molecules, could be used for the delivery of antigens as vaccine adjuvants. The aim of this study was to investigate the influence of (a) composition (fusogenicity and charge) of large multilamellar liposomes, (b) antigen entrapment efficiency into cationic liposomes and (c) addition of immunostimulatory peptidoglycan monomer (PGM) into liposomal formulations on intensity and direction of antigen-specific humoral immune response. Ovalbumin (OVA) was used as a model antigen and liposomal formulations were tested in a well defined experimental mice model. It was shown that, by means of controlling ionic strength of the media, entrapment efficiency of OVA was enhanced and this lead to Th1 biased immune response. Also, by varying liposome composition and increasing fusogenicity of liposomes immune response was directed toward Th1. Addition of immunostimulatory PGM into liposomal formulation resulted in a switch toward Th2 type immune response.     

Synthetic TRP2 long-peptide and α-galactosylceramide formulated into cationic liposomes elicit CD8+ T-cell responses and prevent tumour progression

The lipid antigen α-galactosylceramide (α-GalCer) is a potent activator of invariant natural killer T-cells (iNKT cells) and can stimulate cytotoxic and anti-tumour immune responses. However optimal responses appear to be induced by α-GalCer when cell-based vaccines are delivered intravenously. Here we investigated if co-delivery of protein and peptide antigens along with α-GalCer in a liposomal formulation could stimulate therapeutic anti-tumour immune responses. Cationic liposomes were inherently immune-stimulatory and induced cytotoxic immune responses when delivered both by intravenous and subcutaneous injection. However, only vaccine delivered intravenously stimulated therapeutic anti-tumour immune responses to a peptide antigen. Surface modification with polyethylene glycol (PEG) did not improve immune responses to either intravenously or subcutaneously delivered vaccines. Immune responses to short and long peptide sequences (CD8 and CD4 epitopes) of the self-antigen tyrosinase-related protein 2 (TRP2) as a vaccine antigen, co-delivered with α-GalCer in either cationic liposomes or PBS were further examined. Enhanced production of IFN-γ, increased cytotoxic T-cell responses and tumour survival were observed when a long TRP2-peptide was delivered with α-GalCer in cationic liposomes.     

Mucosal and systemic immune responses by intranasal immunization using archaeal lipid-adjuvanted vaccines

The utility of archaeal polar lipids as an adjuvant/delivery system for elicitation of antigen-specific mucosal immune responses in intranasally administered vaccines was investigated. Although unilamellar archaeosomes (liposomes made from archaeal polar lipids) with encapsulated ovalbumin (OVA/archaeosomes) induced anti-OVA IgG antibody responses in sera, they failed to induce anti-OVA IgA antibody responses at mucosal sites. However, the addition of CaCl2 to convert OVA/archaeosomes into an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) vaccine (OVA/AMVAD) consisting of larger, particulate, aggregated structures resulted in an efficacious intranasal (i.n.) vaccine. Intranasal immunization of mice with OVA/AMVAD vaccines prepared from various archaeal polar lipid compositions elicited anti-OVA IgA antibody responses in sera, feces, bile, vaginal and nasal wash samples. The i.n. immunization also induced anti-OVA IgG, IgG1 and IgG2a antibody responses in sera, as well as cytotoxic T lymphocyte responses. The mucosal and systemic immune responses induced by OVA/AMVAD immunization were generally sustained over several months, and were subject to memory boost responses. Thus, polar archaeal lipids appear to be promising for developing a non-replicating mucosal adjuvant and vaccine delivery system.     

Preparation and preclinical evaluation of experimental group B streptococcus type III polysaccharide-cholera toxin B subunit conjugate vaccine for intranasal immunization

Streptococcus group B (GBS) is usually carried asymptomatically in the vaginal tract of women and can be transferred to the newborn during parturition. Serum antibodies to the capsular polysaccharide (CPS) can prevent invasive diseases, whereas immunity acting at the mucosal surface may be more important to inhibit the mucosal colonization of GBS and thus the risk of infection for the newborn. We prepared different GBS type III CPS-protein conjugate vaccines and evaluated their systemic and mucosal immunogenicity in mice. GBS type III CPS was conjugated to tetanus toxoid (TT) or recombinant cholera toxin B subunit (rCTB) either directly or to rCTB indirectly via TT. The conjugation was performed by different methods: (1) CPS was coupled to TT with 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide (EDAC), using adipic acid dihydrazide (ADH) as a spacer; (2) CPS was conjugated with rCTB using reductive amination; or, (3) N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used to bind rCTB to the TT of the CPS-TT conjugate. Mice were immunized with these conjugates or purified CPS by subcutaneous (s.c.) and intranasal (i. n.) routes. Antibodies to GBS III in serum, lungs and vagina were measured with ELISA. All of the CPS-protein conjugates were superior to unconjugated CPS in eliciting CPS-specific immune responses in serum and mucosal tissue extracts. The conjugates, when administrated s.c., induced only IgG responses in serum, lung and vagina, while i.n. vaccination also elicited IgA responses in the lungs and vagina. The CPS-TT conjugate administrated i.n. induced a strong serum IgG, but only a weak mucosal IgA response, while the CPS-rCTB conjugate elicited high IgG as well as IgA antibodies in the lungs after i.n. immunization. GBS III CPS-TT conjugated with rCTB produced a strong systemic and local anti-CPSIII response after i.n. administration. Co-administration of CT as adjuvant enhanced the anti-CPS systemic and mucosal immune responses further after i.n. administration with the CPS conjugates. These findings indicate that: (i) i.n. immunization with GBS CPS-protein conjugates was more effective than s.c immunization for stimulating serum as well as mucosal immune responses; (ii) rCTB as a carrier protein for GBS III CPS could markedly improve the mucosal immune response; and (iii) the experimental GBS type III CPS conjugates containing rCTB should be investigated as mucosal vaccine to prevent GBS infection in humans.     

A novel recombinant vaccine which protects mice against ricin intoxication

Ricin toxin (RT) is a plant-derived toxin of extraordinary toxicity; a single molecule successfully internalized into the cytoplasm of a cell is lethal for that cell. An estimated dose of 1-10 microg/kg is lethal to humans, making aerosolized ricin a potential agent for bioterrorism. Vaccination against ricin using either denatured toxin or its modified A chain subunit (RTA) has been successful in experimental animals but both vaccines have potential toxicities. Recombinant (r) RTA has not been evaluated as a vaccine. However, the advantage of such a vaccine is that these potential toxicities can be deleted by appropriate mutations. In this study we have generated three mutants and shown that two lack toxicity as compared to the wild type rRTA. These mutants induce protective humoral immune responses in mice. One or both should be considered for use in humans.     

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.     

Dual-route targeted vaccine protects efficiently against botulinum neurotoxin A complex

Clostridium botulinum readily persists in the soil and secretes life-threatening botulinum neurotoxins (BoNTs) that are categorized into serotypes A to H, of which, serotype A (BoNT/A) is the most commonly occurring in nature. An efficacious vaccine with high longevity against BoNT intoxication is urgent. Herein, we developed a dual-route vaccine administered over four consecutive weeks by mucosal and parenteral routes, consisting of the heavy chain (Hc) of BoNT/A targeting dendritic cell peptide (DCpep) expressed by Lactobacillus acidophilus as a secretory immunogenic protein. The administered dual-route vaccine elicited robust and long-lasting memory B cell responses comprising germinal center (GC) B cells and follicular T cells (Tfh) that fully protected mice from lethal oral BoNT/A fatal intoxication. Additionally, passively transferring neutralizing antibodies against BoNT/A into naïve mice induced robust protection against BoNT/A lethal intoxication. Together, a targeted vaccine employing local and systemic administrative routes may represent a novel formulation eliciting protective B cell responses with remarkable longevity against threatening biologic agents such as BoNTs.     

Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine

Bacterial flagellins are potent inducers of innate immune responses in the mouse lung because they bind to TLR5 expressed on the apical surfaces of airway epithelial cells. TLR engagement leads to the initiation of a signaling cascade that results in a pro-inflammatory response with subsequent up-regulation of several cytokines and leads to adaptive immune responses. We examined the ability of two soluble flagellins, a monomeric flagellin expressed in Escherichia coli and a highly purified polymeric flagellin directly isolated from Salmonella, to enhance the efficacy of influenza vaccines in mice. Here we demonstrate that both flagellins co-administered intranasally with inactivated A/PR/8/34 (PR8) virus induced robust increases of systemic influenza-specific IgA and IgG titers and resulted in a more comprehensive humoral response as indicated by the increase of IgG2a and IgG2b subclass responses. Groups immunized with the adjuvanted vaccines were fully protected against high dose lethal challenge by homologous virus whereas inactivated PR8 alone conferred only partial protection. Finally we show that shortly after immunization the adjuvanted vaccines induced a dramatic increase in pro-inflammatory cytokines in the lung, resulting in extensive lung infiltration by granulocytes and monocytes/macrophages. Our results reveal a promising perspective for the use of both soluble monomeric and polymeric flagellin as mucosal vaccine adjuvants to improve protection against influenza epidemics as well as a range of other infectious diseases.     

Enhanced protection against Streptococcus pyogenes infection by intranasal vaccination with a dual antigen component M protein/SfbI lipid core peptide vaccine formulation

We investigated the efficacy of a synthetic Streptococcus pyogenes vaccine targeting two virulence factors using the Lipid Core Peptide (LCP) delivery system. BALB/c mice were immunised intranasally with LCPs containing peptides encompassing T-cell and B-cell epitopes of the conserved C-repeat region of the M protein (J8) or the fibronectin-binding repeats region (FNBR) of SfbI, or a combination formulation containing peptides representing both antigens. LCPs were co-administered with the TLR2/6 agonist MALP-2 as mucosal adjuvant. Humoral and cellular immune responses stimulated at systemic and mucosal levels were strongest in mice immunised with the dual antigen formulation. Mice were completely protected following a respiratory challenge with a lethal dose of a heterologous S. pyogenes strain, whereas there was 70% and 90% survival in mice immunised with LCP-J8 and LCP-FNBR, respectively. This is the first report demonstrating the elicitation of better protective immunity by a dual antigen component S. pyogenes vaccine.