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.     

Monoclonal antibody,mAb 4C13, an effective detoxicant antibody against ricin poisoning

Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20 μg/kg) even at 6 h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin.     

Combination of two candidate subunit vaccine antigens elicits protective immunity to ricin and anthrax toxin in mice

In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population.     

A non-toxic recombinant bivalent chimeric protein rETXm3CSAm4/TMD as a potential vaccine candidate against enterotoxemia and braxy

Clostridium perfringens epsilon toxin (ETX) and Clostridium septicum alpha toxin (CSA) are lethal and necrotizing toxins, which play key roles in enterotoxemia and braxy of ruminants, respectively. In the present study, we synthesized a bivalent chimeric protein rETX_m3CSA_m4/TMD comprising ETX_m3 (Y30A/H106P/Y196A) and CSA_m4/TMD (C86L/N296A/H301A/W342A and a deletion of residues 212 to 222). Compared with recombinant ETX and recombinant CSA, rETX_m3CSA_m4/TMD showed no cytotoxicity in Madin-Darby Canine Kidney cells and was not fatal to mice. Moreover, rETX_m3CSA_m4/TMD could protect immunized mice against 10 × mouse LD₁₀₀ of crude ETX or 3 × mouse LD₁₀₀ of crude CSA without obvious histopathologic difference. Most importantly, both rabbits and sheep immunized with rETX_m3CSA_m4/TMD produced high titers of neutralizing antibody which protected the animals against the challenge with crude ETX or crude CSA. These data suggest that genetically detoxified rETX_m3CSA_m4/TMD is a potential subunit vaccine candidate against enterotoxemia and braxy.     

Development of a hybrid Shiga holotoxoid vaccine to elicit heterologous protection against Shiga toxins types 1 and 2

The hemolytic uremic syndrome is a life-threatening sequela that occurs after infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) or Shigella dysenteriae type 1, and Stx is responsible for initiating this syndrome. An STEC isolate can express Stx1, Stx2, or both, but antisera to Stx1 and Stx2 are not cross-neutralizing. To produce a single vaccine candidate against both toxins, we created a genetic toxoid that contained the enzymatically-inactivated StxA2 subunit and the native StxB1 subunit. We found that mice immunized with this hybrid holotoxoid, developed neutralizing anti-Stx1 and anti-Stx2 antibodies and survived challenge with 10 lethal doses of either or both toxins.     

Passive antibodies derived from intramuscularly immunized toxoid fusion 3xSTaN12S-dmLT protect against STa+ enterotoxigenic Escherichia coli (ETEC) diarrhea in a pig model

Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of children’s diarrhea and travelers’ diarrhea. Developing effective vaccines against ETEC associated diarrhea becomes a top priority. ETEC heat-labile toxin (LT) and heat-stable toxin (STa) toxoid fusion 3xSTa_N12S-dmLT was demonstrated recently to induce neutralizing antitoxin antibodies in intraperitoneally or subcutaneously immunized mice. However, whether antibodies derived from this toxoid fusion are protective against ETEC diarrhea has not been examined. In this study, we intramuscularly immunized pregnant gilts with toxoid fusion 3xSTa_N12S-dmLT, challenged suckling piglets with a STa-positive ETEC strain, and assessed protective efficacy of passive acquire antitoxin antibodies against ETEC diarrhea. Data showed all three immunized gilts developed anti-STa IgG and IgA antibodies, and piglets born to the immunized dams acquired anti-STa and anti-LT antibodies. When challenged with a STa+ ETEC strain, none of the piglets born to the immunized dams developed watery diarrhea, with 20 piglets remained normal and the other 8 piglets developed mild diarrhea indicated with stained butt. In contrast, the control dams and born piglets had no anti-STa or anti-LT antibodies detected, and 26 out 32 piglets developed watery diarrhea after challenge of the STa+ ETEC strain. These results indicated that passive acquired anti-STa antibodies are protective against ETEC diarrhea, and suggested potential application of toxoid fusion 3xSTa_N12S-dmLT in ETEC vaccine development.     

Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against different strains in active and passive challenge models

Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developed countries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A (TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile (B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased virulence is attributed to multiple factors including: increased production of A- and B-toxins; production of binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB₍₀₂₇₎). TcdB₍₀₂₇₎ is more cytotoxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct from historical TcdB (TcdB₍₀₀₃₎). Broadly protective vaccines and therapeutic antibody strategies, therefore, may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficile vaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptor binding domains (RBD) of TcdA, TcdB₍₀₀₃₎, TcdB₍₀₂₇₎ and CDT. Herein, we describe the development of a trivalent toxin (T-toxin) vaccine (CDTb/TcdB₍₀₀₃₎/TcdA) and quadravalent toxin (Q-toxin) vaccine (CDTb/TcB₍₀₀₃₎/TcdA/TcdB₍₀₂₇₎) fusion proteins that retain the protective toxin neutralizing epitopes. Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the generation of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vaccine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype 003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG was produced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies, mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG were completely protected when challenged with historical or epidemic strains of C. difficile. The use of chimeric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeutic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI).     

Potential protective immunogenicity of recombinant Clostridium perfringens α-β2-β1 fusion toxin in mice, sows and cows

Clostridial toxins are main pathogenic virulence of Clostridium perfringens that have been associated with a wide range of diseases in both humans and domestic animals. Genetically engineered toxoids have been shown to function as potential vaccine candidates in the prevention of Clostridium derived infectious diseases. In this study, we have developed recombinant α-toxin (CPA), β2/β1-fusion toxin (CPB2B1) and α/β2/β1 trivalent fusion-toxin (CPAB2B1) as vaccine candidates that may be used to vaccinate against C. perfringens α, β1 and β2-toxins. Mice immunized with these recombinant toxoids demonstrated a strong protective immunological response when administered a lethal dose of C. perfringens type C culture filtrate with high titers of neutralizing antibodies to the toxins in the sera, as well as the intestinal mucosal s-IgA level. Specific neutralizing antibodies to the toxins were also detected in the sera and colostrum of sows and cows vaccinated with the toxoids. Furthermore, the CPA and CPB2B1 recombinant toxoid cocktail was capable of stimulating relatively higher levels of immune responses compared to that of CPA, CPB2B1 and CPAB2B1 alone. The CPAB2B1 trivalent fusion toxoid also displayed increased immunogenicity relative to CPA and CPB2B1 alone. These results suggest that recombinant toxoids are potential vaccine candidates against Clostridial toxins; the use of mixed cocktails and/or multivalent recombinant toxoids against different types of toxins may be an effective approach in the prevention of diseases caused by toxins produced by C. perfringens.     

Plant-based expression of a partially humanized neutralizing monoclonal IgG directed against an immunodominant epitope on the ricin toxin A subunit

GD12 is a murine monoclonal IgG(1) (mAb) that recognizes an immunodominant linear neutralizing epitope (163-TLARSFIICIQM-174) on the A subunit (RTA) of ricin toxin. With the long-term goal of using GD12 as a potential countermeasure against ricin intoxication, we have produced a chimeric derivative of GD12 (cGD12) in which the murine heavy and light chain variable regions were fused to a human IgG(1) framework. The chimeric mAb, expressed and purified using a Nicotiana-based system demonstrated epitope specificity and ricin neutralizing activity similar to the parental murine mAb. Passive administration of cGD12 (10μg) to mice by intraperitoneal injection protected the animals against a systemic ricin challenge. In a post-exposure setting, the murine and chimeric mAbs administered as much as 6h after toxin challenge were each capable of rescuing mice from toxin-induced death, revealing the potential of GD12 to serve as both a prophylactic and therapeutic for ricin intoxication.     

Folding domains within the ricin toxin A subunit as targets of protective antibodies

Efforts to develop an effective vaccine against ricin are focused on the engineering of attenuated and stable recombinant forms of the toxin's enzymatic A subunit (RTA). While several candidate antigens are in development, vaccine design and efficacy studies are being undertaken in the absence of a fundamental understanding of those regions of RTA that are critical in eliciting protective immunity. In this present study, we produced and characterized a collection of monoclonal antibodies (MAbs) directed against five distinct immunodominant regions on RTA, and used these MAbs to identify several key neutralizing epitopes on the toxin. Protective MAbs were directed against α-helices located in RTA folding domains 1 and 2, whereas non-neutralizing antibodies recognized random coils and loops that were primarily confined to folding domain 3. These data offer insights into the immunodominant and structural determinants on RTA that give rise to protective immunity, and for the first time provide an immunological rationale for ricin vaccine design.     

Enhanced immune sera and vaccine: safe approach to treat scorpion envenoming

Irradiation of Androctonus australis hector venom using a dose of 2 kGy has successfully abolished toxicity without reducing its antigenic or immunogenic properties. Toxicity of irradiated antigen was abolished until 20 times of LD₅₀ of native venom. Analysis of physiopathological effects induced by native and irradiated venoms was assessed by the analysis of tissue damage, immunohistochemistry and metabolical analysis in the organs (heart, lungs and liver). Immunological response of Aah venom using native or irradiated venom showed high titers of IgG1 in the plasma of immunized animals with native venom suggesting that Th2 cells were predominantly involved in the immune response. In the other hand, irradiated venom induced high titers of IgG2, indicating a predominantly Th1 type response. A protective effect of immunized mice with irradiated venom was evaluated. Immunized mice were protected from the toxic effects of native venom doses at one, three and six months after immunization. Mice were protected against a challenge of 4 LD₅₀ doses of native venom, one month after immunization. This protective effect was improved and effective at 3 and 6 months, all immunized mice were protected respectively against 6 and 10 LD₅₀ of native venom. At the one-month time point, the protective effect of mice was associated with high levels of antibodies in the plasma of immunized mice. However, despite the persistence of higher protection levels, the antibody titers decreased in a time-dependent manner. These results suggest that additional factors other than circulating antibodies provided the long-term protective activity produced by immunization with irradiated venom.     

Efficacy,heat stability and safety of intranasally administered Bacillus subtilis spore or vegetative cell vaccines expressing tetanus toxin fragment C

Bacillus subtilis strains expressing tetanus toxin fragment C (TTFC) were tested as vaccine candidates against tetanus in adult mice. Mice received three intranasal (IN) exposures to 10⁹ spores or 10⁸ vegetative cells of B. subtilis expressing recombinant TTFC. Immunized mice generated protective systemic and mucosal antibodies and survived challenge with 2× LD₁₀₀ of tetanus toxin. Isotype analysis of serum antibody indicated a balanced Th1/Th2 response. Lyophilized vaccines stored at 45 °C for ≥12 months, remained effective. Immunized conventional and SCID mice remained well, and no histological changes in brain or respiratory tract were detected. Lyophilized/reconstituted B. subtilis tetanus vaccines administered IN to mice appear safe, heat-stable, and protective against lethal tetanus challenge.     

Etx-Y71A as a non-toxic mutant of Clostridium perfringens epsilon toxin induces protective immunity in mice and sheep

Epsilon toxin (Etx) is an extremely potent toxin produced by Clostridium perfringens toxinotypes B and D, which cause fatal enterotoxemia in many livestock species, mainly sheep and goats. Our previous study demonstrated that the aromatic amino acid (AA) residue at position 71 in domain III of Etx is needed for its cytotoxic activity toward MDCK cells. Here, we first determined that Etx mutants with non-aromatic AA substitutions at Tyr71 lost lethality in mice, indicating that the aromatic AA residue at position 71 is a toxicity determinant of Etx in vivo. After intravenous injection with a high dose of the trypsin-activated Etx-Y71A mutant, mice did not show any histopathological lesions, and confocal microscopy observations further showed that Etx-Y71A lost the ability to cross the blood–brain barrier of the mice. These results suggested that the Etx-Y71A mutant is sufficiently safe in vivo to be a vaccine candidate. Furthermore, the immune efficacy of Etx-Y71A was evaluated in model and host animals. Mice inoculated with this mutant produced high levels of neutralizing antibodies and were completely protected from a 100 LD₅₀ of trypsin-activated Etx challenge. Sheep immunized with Etx-Y71A produced high levels of neutralizing antibodies that provided protection in mice against an activated Etx challenge, and lambs could receive passive immunity through immunization of pregnant ewes. Additionally, homology modeling and circular dichroism analysis showed that Etx-Y71A has structural similarity to Etx, which provides a structural basis for Etx-Y71A retaining the immunogenicity of Etx. Taken together, these results suggest that Etx-Y71A is a potential vaccine candidate against Etx-inducing enterotoxemia.     

Heterologous protection against alpha toxins of Clostridium perfringens and Staphylococcus aureus induced by binding domain recombinant chimeric protein

Clostridium perfringens and Staphylococcus aureus are the two important bacteria frequently associated with majority of the soft tissue infections. The severity and progression of the diseases caused by these pathogens are attributed primarily to the alpha toxins they produce. Previously, we synthesized a non-toxic chimeric molecule r-αCS encompassing the binding domains of C. perfringens and S. aureus alpha toxins and demonstrated that the r-αCS hyperimmune polysera reacts with both the native wild type toxins. In the present report, we evaluated efficacy of r-αCS in conferring protection against C. perfringens and S. aureus alpha toxin infections in murine model. Immunization of BALB/c with r-αCS was effective in inducing both high titers of serum anti-r-αCS antibodies after three administrations. Sub-typing the antibody pool revealed high proportions of IgG1 indicating a Th2-polarized immune response. The r-αCS stimulated the proliferation of splenocytes from the immunized mice upon re-induction by the antigen, in vitro. The levels of interleukin-10 increased while TNF-α was found to be downregulated in the r-αCS induced splenocytes. Mice immunized with r-αCS were protected against intramuscular challenge with 5 × LD₁₀₀ doses of C. perfringens and S. aureus alpha toxins with >80% survival, which killed control animals within 48–72 h. Passive immunization of mice with anti-r-αCS serum resulted in 50–80% survival. Our results indicate that r-αCS is a remarkable antigen with protective efficacy against alpha toxin mediated C. perfringens and S. aureus soft tissue co-infections.     

Evaluation of the protective efficacy of recombinant protective antigen vaccine (GC1109)-immunized human sera using passive immunization in a mouse model

The protective efficacy of human sera from vaccinated individuals with a new recombinant protective antigen anthrax vaccine (GC1109) against lethal spore challenge was evaluated in a mouse model. Eighteen human sera were selected from the vaccinated individuals based on their toxin neutralizing assay (TNA) titer (ED₅₀ of 55 to 668). The selected sera were diluted and passively transferred to A/J mice and the mice were subsequently challenged with 100 × LD₅₀ of Bacillus anthracis Sterne spores. The correlation between the survival rate of passively immunized mice and the TNA ED₅₀ of transferred sera was presented (r = 0.873, P-value < 0.001). The estimated TNA titer for 50% survival rate against lethal challenge was 197 (95% confidence interval of 149 and 260). The result suggest that GC1109 is protective against exposure to B. anthracis and the TNA titer of vaccinated serum can be an indicator for protective efficacy.     

A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models

Antibodies targeting the Clostridium difficile toxin A and toxin B confer protective immunity to C. difficile associated disease in animal models and provided protection against recurrent C. difficile disease in human subjects. These antibodies are directed against the receptor binding domains (RBD) located in the carboxy-terminal portion of both toxins and inhibit binding of the toxins to their receptors. We have constructed a recombinant fusion protein containing portions of the RBD from both toxin A and toxin B and expressed it in Escherichia coli. The fusion protein induced high levels of serum antibodies to both toxins A and B capable of neutralizing toxin activity both in vitro and in vivo. In a hamster C. difficile infection model, immunization with the fusion protein reduced disease severity and conferred significant protection against a lethal dose of C. difficile spores. Our studies demonstrate the potential of the fusion protein as a vaccine that could provide protection from C. difficile disease in humans.     

Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E. coli heat-labile enterotoxin B subunit

Peptides KDYGASTGSSL (Epil). SLLRRRRNGEDVSV (Epi3) and DDEIYGNDGHP (Epi6), predicted to constitute immunogenic epitopes of the hemolysin-cytotoxin ApxIA of Actinobacillus pleuropneumoniae were inserted into a surface-exposed loop of the B subunit of the E. coli heat-labile enterotoxin (EtxB). The resulting chimeric proteins were recognized by monospecific antibodies against purified native ApxI and by convalescent sera of pigs that were positive for A. pleuropneumoniae serotype 1. Mice anti-sera against chimeric proteins EtxB::ApxIAEpi3 and EtxB::ApxIAEpi6 reacted with purified ApxI. These results indicate that Epi3 and Epi6 regions constitute linear epitopes of the structural ApxIA protein toxin. Epitope Epi6 which is located in the structure of the glycine rich repeats in ApxI elicits the formation of hemolysin neutralizing antibodies when introduced into mice in the form of a chimeric EtxB fusion protein. We suggest that fusion of peptide sequences to EtxB is a useful tool for the analysis of epitopes of complex proteins such as RTX toxins.     

Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax^TM vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum + CpG. We also evaluated mice immunized with RBD/alum + CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum + CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum + CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2, including variants of concern.     

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.     

Chimeric hepatitis B virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against Bacillus anthracis

The major aim of present study is to develop and evaluate chimeric virus-like particles (VLPs) displaying a neutralizing epitope of anthrax protective antigen (PA) as a potential vaccine against anthrax. The truncated hepatitis B virus core (HBc) protein (aa 1-144) was used as a carrier, and the 2beta2-2beta3 loop of the PA domain 2 (aa 302-325) which has been shown contains a dominant neutralizing epitope was inserted into the major immunodominant region (MIR) of the HBc. The recombinant protein HBc-N144-PA-loop2 was expressed in Escherichia coli, and was able to form HBc-like particles confirmed by electron microscopy. The immunogenicity of these chimeric particles was evaluated in mice and guinea pigs. In mice the HBc-N144-PA-loop2 was able to induce PA-epitope specific antibodies; in guinea pigs it was able to induce PA-epitope specific antibodies and anthrax toxin-neutralizing antibodies regardless of whether alum adjuvant was used or not, and was able to partially protect the immunized guinea pigs against virulent anthrax spores challenge. This study suggests chimeric HBc particles carrying a neutralizing epitope of PA can induce protective immunity against Bacillus anthracis.