Efficacy and Immunogenicity of Single-Dose AdVAV Intranasal Anthrax Vaccine Compared to Anthrax Vaccine Absorbed in an Aerosolized Spore Rabbit Challenge Model

AdVAV is a replication-deficient adenovirus type 5-vectored vaccine expressing the 83-kDa protective antigen (PA83) from Bacillus anthracis that is being developed for the prevention of disease caused by inhalation of aerosolized B. anthracis spores. A noninferiority study comparing the efficacy of AdVAV to the currently licensed Anthrax Vaccine Absorbed (AVA; BioThrax) was performed in New Zealand White rabbits using postchallenge survival as the study endpoint (20% noninferiority margin for survival). Three groups of 32 rabbits were vaccinated with a single intranasal dose of AdVAV (7.5 × 10⁷, 1.5 × 10⁹, or 3.5 × 10¹⁰ viral particles). Three additional groups of 32 animals received two doses of either intranasal AdVAV (3.5 × 10¹⁰ viral particles) or intramuscular AVA (diluted 1:16 or 1:64) 28 days apart. The placebo group of 16 rabbits received a single intranasal dose of AdVAV formulation buffer. All animals were challenged via the inhalation route with a targeted dose of 200 times the 50% lethal dose (LD₅₀) of aerosolized B. anthracis Ames spores 70 days after the initial vaccination and were followed for 3 weeks. PA83 immunogenicity was evaluated by validated toxin neutralizing antibody and serum anti-PA83 IgG enzyme-linked immunosorbent assays (ELISAs). All animals in the placebo cohort died from the challenge. Three of the four AdVAV dose cohorts tested, including two single-dose cohorts, achieved statistical noninferiority relative to the AVA comparator group, with survival rates between 97% and 100%. Vaccination with AdVAV also produced antibody titers with earlier onset and greater persistence than vaccination with AVA.     

Human Leukocyte Antigens and Cellular Immune Responses to Anthrax Vaccine Adsorbed

Interindividual variations in vaccine-induced immune responses are in part due to host genetic polymorphisms in the human leukocyte antigen (HLA) and other gene families. This study examined associations between HLA genotypes, haplotypes, and homozygosity and protective antigen (PA)-specific cellular immune responses in healthy subjects following immunization with Anthrax Vaccine Adsorbed (AVA). While limited associations were observed between individual HLA alleles or haplotypes and variable lymphocyte proliferative (LP) responses to AVA, analyses of homozygosity supported the hypothesis of a “heterozygote advantage.” Individuals who were homozygous for any HLA locus demonstrated significantly lower PA-specific LP than subjects who were heterozygous at all eight loci (median stimulation indices [SI], 1.84 versus 2.95, P = 0.009). Similarly, we found that class I (HLA-A) and class II (HLA-DQA1 and HLA-DQB1) homozygosity was significantly associated with an overall decrease in LP compared with heterozygosity at those three loci. Specifically, individuals who were homozygous at these loci had significantly lower PA-specific LP than subjects heterozygous for HLA-A (median SI, 1.48 versus 2.13, P = 0.005), HLA-DQA1 (median SI, 1.75 versus 2.11, P = 0.007), and HLA-DQB1 (median SI, 1.48 versus 2.13, P = 0.002) loci, respectively. Finally, homozygosity at an increasing number (≥4) of HLA loci was significantly correlated with a reduction in LP response (P < 0.001) in a dose-dependent manner. Additional studies are needed to reproduce these findings and determine whether HLA-heterozygous individuals generate stronger cellular immune response to other virulence factors (Bacillus anthracis LF and EF) than HLA-homozygous subjects.     

Promising Rabies Vaccine for Postexposure Prophylaxis in Developing Countries,a Purified Vero Cell Vaccine Produced in China

We evaluated the immunogenicity, safety, and antibody persistence of a Vero cell rabies vaccine manufactured in China, compared with those of Verorab. Adequate titers of antibody were observed for the two vaccines. ChengDa rabies vaccine could be a promising alternative vaccine for many developing countries which cannot afford expensive rabies vaccines.Although effective rabies vaccines for postexposure treatment are available (3), there are still about 50,000 to 60,000 human deaths annually. China, as the largest developing country in the world, has endeavored tremendously in rabies prevention and vaccine manufacturing. China accounts for almost two-thirds of the total rabies vaccines used in Asia (9), with the locally produced tissue culture vaccine being safe and relatively inexpensive (5). Currently, the most common rabies vaccine used in China is purified Vero cell vaccine, referred to as ChengDa (Liaoning ChengDa Biological Co., Ltd., Shengyang, China). ChengDa rabies vaccine is grown on a Vero cell line utilizing the L. Pasteur 2061 strain of rabies virus. It is inactivated with β-propiolactone (BPL), lyophilized, and reconstituted in 0.5 ml of physiological saline. It is manufactured according to good manufacturing practices (GMP) and strictly fulfills the WHO recommendations for potency (8). ChengDa was licensed by the Health Ministry of China and the State Food and Drug Administration of China (SFDA) in 2002 and has been marketed throughout the country since that time.This study was designed to compare the immunogenicity, safety, and persistence of the two different Vero cell rabies vaccines (ChengDa and Verorab) available in China. The study was conducted in the Emergency Department, Beijing University People''s Hospital, and the antibody evaluation was performed primarily at the National Institute for the Control of Pharmaceutical and Biological Products. Formal ethical approval was obtained from the ethics committee of Beijing University People''s Hospital. Written informed consent was obtained from each subject and/or his/her legal representative. Intramuscular doses of either 0.5 ml of ChengDa or 0.5 ml of Verorab were given to 500 patients on days 0, 3, 7, 14, and 28. Blood samples from all vaccinees were taken on days 0, 7, 14, 45, and 365. Antibody testing was performed using the rapid fluorescent focus inhibition test (RFFIT) in 96-well microplates according to the technique of Zalan et al. (10).From February 2006 to September 2008, a total of 500 patients with WHO-designated category I or II rabies exposure were enrolled and randomly assigned to the ChengDa group or the Verorab group. A study flow chart is shown in Fig. ​Fig.1.1. The baseline demographic characteristics are demonstrated in Table ​Table1,1, and subjects were similarly distributed in the two groups. The exclusion criteria included poor compliance, primary or acquired immunodeficiency, corticosteroid intake, and previous rabies immunization. Rabid patients and patients who were simultaneously in other clinical trials were also excluded.Open in a separate windowFIG. 1.Study flow chart.

TABLE 1.

Baseline demographic characteristics of subjects

Comprehensive Analysis and Selection of Anthrax Vaccine Adsorbed Immune Correlates of Protection in Rhesus Macaques

Humoral and cell-mediated immune correlates of protection (COP) for inhalation anthrax in a rhesus macaque (Macaca mulatta) model were determined. The immunological and survival data were from 114 vaccinated and 23 control animals exposed to Bacillus anthracis spores at 12, 30, or 52 months after the first vaccination. The vaccinated animals received a 3-dose intramuscular priming series (3-i.m.) of anthrax vaccine adsorbed (AVA) (BioThrax) at 0, 1, and 6 months. The immune responses were modulated by administering a range of vaccine dilutions. Together with the vaccine dilution dose and interval between the first vaccination and challenge, each of 80 immune response variables to anthrax toxin protective antigen (PA) at every available study time point was analyzed as a potential COP by logistic regression penalized by least absolute shrinkage and selection operator (LASSO) or elastic net. The anti-PA IgG level at the last available time point before challenge (last) and lymphocyte stimulation index (SI) at months 2 and 6 were identified consistently as a COP. Anti-PA IgG levels and lethal toxin neutralization activity (TNA) at months 6 and 7 (peak) and the frequency of gamma interferon (IFN-γ)-secreting cells at month 6 also had statistically significant positive correlations with survival. The ratio of interleukin 4 (IL-4) mRNA to IFN-γ mRNA at month 6 also had a statistically significant negative correlation with survival. TNA had lower accuracy as a COP than did anti-PA IgG response. Following the 3-i.m. priming with AVA, the anti-PA IgG responses at the time of exposure or at month 7 were practicable and accurate metrics for correlating vaccine-induced immunity with protection against inhalation anthrax.     

Anthrax Spores Make an Essential Contribution to Vaccine Efficacy

Anthrax is caused by Bacillus anthracis, a gram-positive spore-forming bacterium. Septicemia and toxemia rapidly lead to death in infected mammal hosts. Currently used acellular vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, in experimental animals such vaccines are less protective than live attenuated strains. Here we demonstrate that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA elicits total protection against challenge with virulent B. anthracis strains in mice and guinea pigs. The toxin-neutralizing activities of sera from mice immunized with PA alone or PA plus FIS were similar, suggesting that the protection conferred by PA plus FIS was not only a consequence of the humoral response to PA. A PA-deficient challenge strain was constructed, and its virulence was due solely to its multiplication. Immunization with FIS alone was sufficient to protect mice partially, and guinea pigs totally, against infection with this strain. This suggests that spore antigens contribute to protection. Guinea pigs and mice had very different susceptibilities to infection with the nontoxigenic strain, highlighting the importance of verifying the pertinence of animal models for evaluating anthrax vaccines.     

Humoral and Cell-Mediated Immune Responses to Alternate Booster Schedules of Anthrax Vaccine Adsorbed in Humans

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7, r² = 0.86, P < 0.0001, log₁₀ transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00119067","term_id":"NCT00119067"}}NCT00119067.)     

Frequency and Domain Specificity of Toxin-Neutralizing Paratopes in the Human Antibody Response to Anthrax Vaccine Adsorbed

Protective antigen (PA) is the cell surface recognition unit of the binary anthrax toxin system and the primary immunogenic component in both the current and proposed “next-generation” anthrax vaccines. Several studies utilizing animal models have indicated that PA-specific antibodies, acquired by either active or passive immunization, are sufficient to protect against infection with Bacillus anthracis. To investigate the human antibody response to anthrax immunization, we have established a large panel of human PA-specific monoclonal antibodies derived from multiple individuals vaccinated with the currently approved anthrax vaccine BioThrax. We have determined that although these antibodies bind PA in standard binding assays such as enzyme-linked immunosorbent assay, Western blotting, capture assays, and dot blots, less than 25% are capable of neutralizing lethal toxin (LT) in vitro. Nonneutralizing antibodies also fail to neutralize toxin when present in combination with other nonneutralizing paratopes. Although neutralizing antibodies recognize determinants throughout the PA monomer, they are significantly less common among those paratopes that bind to the immunodominant amino-terminal portion of the molecule. These findings demonstrate that PA binding alone is not sufficient to neutralize LT and suggest that for an antibody to effectively block PA-mediated toxicity, it must bind to PA such that one of the requisite toxin functions is disrupted. A vaccine design strategy that directed a higher percentage of the antibody response toward neutralizing epitopes may result in a more efficacious vaccine for the prevention of anthrax infection.The Bacillus anthracis binary toxin system contributes directly to anthrax pathogenicity in the host (3, 14). The cell surface recognition element of this toxin system is an 83-kDa protein known as protective antigen (PA₈₃). Antibodies that bind PA protect against infection (8, 12), and PA is the primary immunogenic component in the anthrax vaccine currently licensed for use in the United States (BioThrax, or anthrax vaccine adsorbed [AVA]; Emergent Biosystems). Ongoing attempts to develop a “next-generation” anthrax vaccine are relying on a recombinant form of PA as the sole immunogenic component. PA''s role as an important vaccine target has driven a significant amount of research into both the biology of this protein toxin and the immunobiology of its interaction with the immune system of the vaccinated or infected host.PA₈₃ binds to the cell surface receptors tumor endothelial marker 8 and the capillary morphogenesis gene 2 product (4, 20). Bound PA is cleaved by cell surface-associated furin proteases to release the 20-kDa amino-terminal portion of the molecule (PA₂₀), which has no further role in intoxication. Following proteolytic cleavage, cell-bound PA₆₃ self-assembles to form a heptameric prepore structure that can bind several molecules of the catalytic toxin components lethal factor (LF) and/or edema factor (EF). Receptor-mediated endocytosis results in the internalization of the complex, which inserts into the membrane of the endocytic vacuole. LF and/or EF is then actively translocated into the cytoplasm of the cell. The structure of PA, both as a monomer and as a heptamer, has been determined (15, 19), and the regions of the molecule (domains) involved in the various functions described above have been identified (1, 6, 15, 18, 19).The immunobiology of the immune response to PA in vaccinated humans has only recently been explored at the molecular level. PA elicits a polyclonal antibody response in vaccinated humans that utilizes a wide variety of immunoglobulin variable (V)-region genes. Preliminary studies have indicated that after vaccination, antibodies undergo the somatic hypermutation and class switch normally associated with affinity maturation (21). We have previously demonstrated the human antibody response to PA to be significantly biased toward epitopes associated with the amino-terminal domain of the PA protein (PA₂₀) and have postulated that these antibodies may be deficient in their ability to neutralize toxin (16).In this study, we determined the toxin neutralization potentials of a large panel of individual monoclonal antibodies isolated from seven individuals vaccinated with AVA vaccine, using a cell-based assay of LT-mediated cytotoxicity. We found that only 24% of the component antibodies that comprise the overall response are capable of neutralizing PA-mediated cytotoxicity in vitro. We found no direct correlation between the relative PA binding ability of the individual antibodies and their ability to neutralize anthrax toxin. We also determined that toxin-neutralizing paratopes occur less frequently among those antibodies that recognize the immunodominant epitopes associated with the amino-terminal domain of the PA monomer. These findings suggest that the efficacy of future PA-based vaccines might be improved by modifying the immunogen such that a greater proportion of the antibody response is directed toward those epitopes that lead to toxin neutralization.     

Development of an Aotus nancymaae Model for Shigella Vaccine Immunogenicity and Efficacy Studies

Several animal models exist to evaluate the immunogenicity and protective efficacy of candidate Shigella vaccines. The two most widely used nonprimate models for vaccine development include a murine pulmonary challenge model and a guinea pig keratoconjunctivitis model. Nonhuman primate models exhibit clinical features and gross and microscopic colonic lesions that mimic those induced in human shigellosis. Challenge models for enterotoxigenic Escherichia coli (ETEC) and Campylobacter spp. have been successfully developed with Aotus nancymaae, and the addition of a Shigella-Aotus challenge model would facilitate the testing of combination vaccines. A series of experiments were designed to identify the dose of Shigella flexneri 2a strain 2457T that induces an attack rate of 75% in the Aotus monkey. After primary challenge, the dose required to induce an attack rate of 75% was calculated to be 1 × 10¹¹ CFU. Shigella-specific immune responses were low after primary challenge and subsequently boosted upon rechallenge. However, preexisting immunity derived from the primary challenge was insufficient to protect against the homologous Shigella serotype. A successive study in A. nancymaae evaluated the ability of multiple oral immunizations with live-attenuated Shigella vaccine strain SC602 to protect against challenge. After three oral immunizations, animals were challenged with S. flexneri 2a 2457T. A 70% attack rate was demonstrated in control animals, whereas animals immunized with vaccine strain SC602 were protected from challenge (efficacy of 80%; P = 0.05). The overall study results indicate that the Shigella-Aotus nancymaae challenge model may be a valuable tool for evaluating vaccine efficacy and investigating immune correlates of protection.     

Vaccination of Rhesus Macaques with the Anthrax Vaccine Adsorbed Vaccine Produces a Serum Antibody Response That Effectively Neutralizes Receptor-Bound Protective Antigen In Vitro

Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.Anthrax is caused by infection with Bacillus anthracis, and its pathogenesis is associated with an antiphagocytic poly-d-glutamic acid capsule and a binary anthrax toxin (ATx). The ATx comprises two protein exotoxins: lethal toxin (LTx) and edema toxin (ETx). The two toxins both have a binding protein called protective antigen (PA) but have separate effector proteins, edema factor (EF) and lethal factor (LF) (3, 20, 56). LTx is composed of PA and LF, and ETx is composed of PA and EF. In the initial stages of infection by B. anthracis, full-length 83-kDa PA (PA83) secreted by the bacterium binds to either one or both of at least two host cell ATx receptors (ATRs): tumor endothelial marker 8 (TEM8) (7, 27, 57) or capillary morphogenesis protein 2 (CMG2) (51).Vaccines containing PA as the major component confer protective efficacy in various animal models of multiple routes of infection (5, 14-16, 29, 42-44, 59). PA has four domains: an amino-terminal domain (domain 1, which is composed of subregions 1a and 1b) that contains two calcium ions and the S₁₆₃RKKRS₁₆₈ cleavage site for activating proteases, a heptamerization domain (domain 2) that contains a large flexible loop implicated in membrane insertion, a small domain (domain 3) hypothesized to aid in oligomerization, and a carboxy-terminal receptor-binding domain (domain 4) (26, 41). Upon binding to an ATR, PA is proteolytically cleaved by the cell surface protease furin to a 63-kDa polypeptide (PA63), releasing a 20-kDa amino-terminal fragment (domain 1a). Cleavage and release of domain 1a facilitate assembly of the PA prepore; a heptameric ring-shaped structure with a negatively charged lumen. Assembly of the prepore exposes a large hydrophobic surface for binding of LF and/or EF molecules to form ATx (9, 17, 19, 26, 33, 35, 37, 41). One PA63 heptamer is able to bind up to three LF and/or EF molecules. The ATx is then endocytosed by a lipid raft-mediated clathrin-dependent process (1, 34). The low-pH conditions (pH 5.5) in the endosome induce the prepore to undergo a conformational switch that translocates ATx to the target cell cytosol (6, 17, 18, 24, 32, 37, 41, 55).The current licensed vaccine for use in humans is anthrax vaccine adsorbed (AVA [BioThrax]; Emergent BioSolutions, Lansing, MI). AVA is a cell-free filtrate from a toxigenic, nonencapsulated B. anthracis strain, V770-NP1-R (2, 10, 45). The primary immunogen is PA (59) adsorbed to aluminum hydroxide adjuvant (10, 29). The current AVA vaccination schedule consists of five 0.5-ml intramuscular (i.m.) injections at 0 and 4 weeks and 6, 12, and 18 months, with annual boosters (10, 30).There are various potential molecular targets in which the host humoral antibody response to vaccination with AVA or PA can interfere with ATx-mediated cytotoxicity. These targets include, but are not limited to, (i) blocking of free PA83 binding to the host cell ATx receptor (TEM8 or CMG2); (ii) inhibition of PA83 proteolytic cleavage by the host cell surface furin-like enzyme or serum proteases, leaving the PA unprocessed and thus unable to form toxin complexes; (iii) interruption of PA63 heptamerization to form the prepore on the host cell surface; (iv) blocking the binding of LF and EF monomers to the PA heptamer prepore; and (v) disruption of internalization and translocation of the ATx. Consequently, PA has become a focal point in developing immunotherapies and next-generation vaccines for the prevention and treatment of anthrax (4, 13, 21, 22, 31, 36, 39, 40, 53, 58, 60).Most of the anti-PA therapies under development specifically target PA domains 2 and 4, with domain 4 being the most frequent target (21, 53, 60). The therapeutic effects of antibodies targeted against domain 4 are considered to be based primarily on blocking the interaction of PA with its host cell receptor (26, 49). However, in active immunization, there will be multiple epitopes presented to the host immune system that are critical to mounting a protective immune response and, likewise, others that may make little or no contribution. Although PA20 is cleaved from PA83 and has no described role in the intoxication process, recent reports have proposed that in AVA-vaccinated humans, the PA20 fragment (domain 1a) contains immunodominant epitopes (48, 61). Therefore, it was postulated that vaccines containing full-length PA (PA83) may be suboptimal due to the dominance of PA20 and that perhaps PA63-based vaccines may be more advantageous (47, 48).To address the question of suboptimal immune responses in PA83-based vaccine and therapeutic design, we developed two low-temperature anthrax lethal toxin (LTx) neutralization activity (TNA) assays, the noncomplexed TNA (NC-TNA) and receptor-bound TNA (RB-TNA) assays. These assays allow comparison of antibody-mediated neutralization of LTx both before and after receptor binding by PA. The goal of this work was to evaluate the ability of anti-PA antibody responses in AVA-vaccinated and inhalation anthrax-challenged rhesus macaques (Macaca mulatta) to neutralize anthrax LTx in vitro both before and after PA has bound to, and been processed at, the cell surface receptor.     

Efficacy of a Vaccine Based on Protective Antigen and Killed Spores against Experimental Inhalational Anthrax

Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD₅₀) and against an aerosol with 75 LD₅₀ of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.Bacillus anthracis is a gram-positive, aerobic, facultatively anaerobic, spore-forming, rod-shaped bacterium and is the etiologic agent of anthrax. B. anthracis resides in the soil as a dormant spore that is highly resistant to adverse conditions and can remain viable for years. The spore typically enters herbivores through ingestion; although anthrax is predominantly a disease of herbivores, humans can be infected through incidental exposure during handling of animals or animal products. In humans, the disease may take three forms—cutaneous, gastrointestinal, or pulmonary—depending on the site of entry. The most common human form is cutaneous anthrax, typically caused by spores infecting open wounds or skin abrasions. The mortality of cutaneous anthrax is near 20% if untreated (21). Gastrointestinal anthrax may in some cases extend to neuromeningitidis and generally leads to fatal systemic disease if untreated (5, 21). Naturally acquired pulmonary anthrax is very unusual. However, the mortality of pulmonary anthrax is almost 100% if not treated very early (80). Inhalational anthrax manifests as the rapid development of nonspecific, flulike symptoms that, if untreated, progress quickly to shock, respiratory distress, and death (21, 80).Inhaled spores are deposited in alveolar spaces where they are ingested by macrophages (39, 66) and by dendritic cells (DCs) (9, 15). Then, the intracellular spores germinate into nascent bacilli that escape from the macrophage, multiply extracellularly in the lymphatic system and spread into the bloodstream, where rapid multiplication continues (38, 39); alternatively, phagocytized spores are transported by migrating macrophages to the mediastinal and peribronchial lymph nodes, where they germinate into bacilli (66). DCs may be central to this step of the infection (15). Anthrax disease appears to result from a two-step process involving overwhelming bacterial replication and subsequent toxin production. Nevertheless, the fate of spores within macrophages, the resistance of macrophages to anthrax toxins and the role of macrophages in B. anthracis dissemination all remain controversial (19, 20, 38, 39, 83). An alternative mechanism has been recently described, suggesting that inhaled spores establish an initial infection in nasally associated lymphoid tissues where they germinate. The bacteria then disseminate first to the draining lymph nodes, then to the spleen and lungs, and finally to the blood (37).B. anthracis has two major virulence determinants. One is a tripartite protein complex toxin composed of lethal factor (LF), edema factor (EF), and protective antigen (PA) all encoded by plasmid pXO1. The other is antiphagocytic poly-γ-d-glutamic acid (γPDGA) capsule encoded by plasmid pXO2. EF and LF combine with PA to form the edema toxin (ET) and lethal toxin (LT), respectively, which both impair host immune defenses and probably act synergistically in vivo to cause edema formation and death (58, 75). The PA-LF/PA-EF complex is internalized by receptor-mediated endocytosis and, after acidification of the endosome, the toxin is translocated into the host cell cytosol, where it exerts cytotoxic effects (89). LT is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinases, leading to toxic effects on susceptible macrophages (3, 18, 24, 54) and impairment of the bactericidal activity of alveolar macrophages, thus facilitating B. anthracis survival (35, 65). ET is a calmodulin-dependent adenylate cyclase that catalyzes the production of cyclic AMP from host ATP, perturbing water homeostasis, which in turn causes massive edema (55). ET is also cytotoxic in a cell-dependent manner and may contribute to the disease through directly killing cells, leading to tissue necrosis (79) and multiorgan failure, resulting in host death (28). LT and ET cooperatively inhibit activation of both DCs (14, 76) and T cells (57), thereby suppressing both the innate immune response and the priming of adaptive immune responses. Therefore, preventing either the entry of the toxin complex into the host cell or its translocation into the cytosol would make a major contribution to protection.The PDGA capsule is a poorly immunogenic polypeptide but seems to be vital for the dissemination of B. anthracis in the bodies of infected animals (12). The in vivo synthesis of capsule determines the outcome of infection (22, 49), and capsule degradation enhances both in vitro macrophage phagocytosis and neutrophil killing of encapsulated B. anthracis (68).The potential use of B. anthracis spores as a weapon of biological warfare or as inhaled weapons of bioterrorism has increased the need for a safe and effective vaccine to protect humans against inhalational anthrax (6, 31).The current United Kingdom licensed anthrax vaccine, anthrax vaccine precipitate, is an alum-precipitated filtrate of B. anthracis 34F2 Sterne strain culture consisting mainly of PA (77). The U.S. licensed anthrax vaccine absorbed (AVA/Biothrax) also consists mainly of PA, in this case extracted from cultures of the unencapsulated, toxin-producing strain of B. anthracis V770-NP1-R adsorbed onto aluminum hydroxide (33). Both vaccines contain small amounts of EF and LF and probably other components that presumably contribute to vaccine efficacy (33, 77, 88).These vaccines have the major disadvantage of inducing only a limited duration of protection and require frequent booster injections if sufficient immunity is to be maintained (32). Furthermore, such PA-based vaccines, acting on toxins, are less effective than live attenuated vaccines such as the Sterne strain, suggesting that additional antigens may contribute in a significant manner to protective immunity (4, 16, 42, 51, 59, 85).Various animal models have been used for testing the protective activities of vaccines against anthrax infection, including mice (10, 30, 86), rats (46), guinea pigs (10, 26, 46, 70), hamsters (27), rabbits (26, 50, 60, 61), and nonhuman primates (26, 40, 44, 60). These studies emphasize the large differences of protection between species. For instance, PA-based vaccines confer better protection to guinea pigs, rabbits, and nonhuman primates than to mice, probably because the γPDGA capsule is the primary virulence factor in mice (87). Indeed, many reports suggest that capsule antigen(s) (13, 47, 64, 67, 81) and spore antigen(s) (10, 16, 23) might confer additional protection. An immunodominant glycoprotein antigen of the spore surface (BclA) has been identified among the various surface proteins of the exosporium and may contribute to protective immunity (72, 74). Sera from animals immunized with living spores of the toxinogenic unencapsulated STI-1 strain of B. anthracis have been reported to express both antitoxin and antispore activities, the latter involving inhibition of spore germination, which was attributed by some authors to both anti-PA and anti-LF antibodies (73). Furthermore, PA-based vaccines induce antispore activity characterized by stimulation of phagocytosis of opsonized spores by murine macrophages in vitro and by inhibition of spore germination. As a consequence, anti-PA antibody-specific immunity may contribute to impeding the early stages of infection with B. anthracis spores (84).Brossier et al. demonstrated that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA antigen (PA-FIS) elicits total protection of mice and guinea pigs against subcutaneous (s.c.) challenge with a virulent B. anthracis strain (10). However, vaccines that are effective for the s.c. route of infection may not be so against the pulmonary route (30).Several studies have demonstrated that either live spore-based vaccines or PA-based vaccines may confer variable protection against different B. anthracis strains and isolates in both mice and guinea pigs (26, 43, 51, 82, 85). Therefore, we used two different B. anthracis challenge strains in our study, namely, strains 9602 and 17JB from the Institut Pasteur collection. Although both strains are encapsulated and toxinogenic (cap⁺ tox⁺), harboring both pXO1 and pXO2 plasmids, they differ in virulence, as shown by the 50% lethal doses (LD₅₀) (s.c. route), estimated to be about 50 and 500 spores per mouse, respectively (10). Strain 9602 is as virulent as the Ames strain (10, 43); strain 17JB (the atypical Pasteur vaccine strain 2-17JB (78), harboring both pXO1 and pXO2 (cap+ tox+), is very similar to the so-called “Carbosap” strain used in Italy for immunization against ovine and bovine anthrax (25). It has residual pathogenicity characteristics that cause death in mice and guinea pigs but expresses no virulence in rabbits (25). Adone et al. demonstrated that the attenuation of the Carbosap vaccine strain is not due to the lack of virulence genes (cya, lef, and pagA), of regulatory genes (atxA and pagR), or of the gerX operon involved in germination within macrophages, or to divergence of the sequences of these genes from those of a wild-type virulent B. anthracis strain (1). Indeed, sophisticated advanced molecular analysis has been unable to identify the genetic differences accounting for differences in virulence between Carbosap and virulent strains (48).There are various possible causes of these differences in virulence and pathogenesis, including (i) involvement of unknown virulence factors and/or mechanisms involved in attenuation, (ii) differences in expression and activity of the known virulence factors and their regulators (48), and (iii) differences in pXO2 plasmid copy number (17). Nevertheless, like the Vollum strain, 17JB remains a relevant model for the study of vaccine efficacy: it is less pathogenic than wild-type strains such as 9602 or Ames but is nevertheless cap⁺ tox⁺.In summary, despite obvious efficacy in nonhuman primates, the currently licensed anthrax vaccines have shortcomings, such as a limited duration of protection and the need for frequent booster injections. Moreover, trace amounts of LF, EF, and probably other components are likely to have contributed to the efficacy of the vaccine in the reported studies. For instance, AVA provides partial protection in a guinea pig model of inhalational anthrax, whereas a recombinant anthrax PA from B. anthracis (rPA)-based vaccine elicits no protection (53). Furthermore, PA-based vaccines may confer variable protection against different B. anthracis strains and isolates, and large differences in the level of protection afforded are observed between animal species. These limitations have stimulated interest in the development of improved anthrax vaccines. The data discussed above suggest that other antigens in addition to PA are required for full protection.The aim of the present study was to optimize the PA-FIS vaccine immunization protocol so as to elicit protection against inhalational anthrax in an experimental model of lung infection. We assessed the systemic and mucosal immune response to PA-FIS in mice and guinea pigs, immunized either through the s.c. or the intranasal (i.n.) route or both. Second, we assessed the protection afforded in an experimental model of inhalational anthrax of mice and guinea pigs infected by nasal instillation or an aerosol.     

A Three-Dose Intramuscular Injection Schedule of Anthrax Vaccine Adsorbed Generates Sustained Humoral and Cellular Immune Responses to Protective Antigen and Provides Long-Term Protection against Inhalation Anthrax in Rhesus Macaques

A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r² = 0.89 for log₁₀-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4⁺ cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.     

Immunogenicity and Protective Efficacy of a Recombinant Subunit West Nile Virus Vaccine in Rhesus Monkeys

The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques (Macaca mulatta). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 μg of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzyme-linked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 μg of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or 25-μg dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.West Nile virus (WNV) was first detected in North America in 1999 and spread rapidly across the continental United States (3, 32), as well as into Canada (8), Mexico (9), and Central and South America (17). The virus is transmitted via mosquitoes, primarily through the bite of Culex species but also by many other genera of mosquitoes (14). Birds are the natural hosts and serve as the zoonotic reservoir, while mammals and reptiles are considered to be incidental hosts from which, it is believed, further transmission generally does not occur (12). This is thought to be due to the relatively low levels of viremia that develop in these latter hosts, which may be insufficient to allow for secondary mosquito transmission (11). However, more recent studies (2, 38) have suggested that, in some mammals and reptiles, sufficient viremia may develop to yield at least a low competence for transmission.Based on retrospective seroepidemiological surveys conducted after the initial discovery of this virus in North America, it was determined that about 20% of those individuals infected developed clinical disease (3). The large majority of clinical cases resulted in a self-limiting, influenza-like syndrome (3); however, about 1 in 150 infected patients developed neurological complications (28). These complications included cases of meningitis; encephalitis; meningoencephalitis; and an acute, flaccid paralytic, poliomyelitis-like syndrome (13). The cases with WNV-associated neurological complications tend to be severe, often resulting in permanent disabilities, with reported case fatality rates of 5 to 15% (3). However, in a recent study (4), it was reported that even in those cases of mild, nonneuropathological disease, after resolution of the infection had apparently occurred, residual defects in neuromotor and cognitive function could be measured for at least 1 year after the original diagnosis.Moreover, the disease course tends to be much more severe in elderly individuals, with significantly higher case fatality rates of about 30% in neuroinvasive cases (5, 30, 33, 42). This may be due to declining immunocompetence concomitant with aging (“immunosenescence”). In addition to the elderly population, individuals whose immune systems have been compromised through primary immune deficiencies, acquired deficiencies, or immunosuppressive therapies are also at increased risk of severe disease caused by WNV infection (10, 18). Certain other chronic diseases, such as diabetes mellitus and hypertension, may also render individuals infected with WNV more susceptible to developing severe disease (16).WNV is a member of the Flaviviridae family, genus Flavivirus. It is an enveloped, positive-strand RNA virus. The RNA genome comprises 10 genes, coding for three structural and seven nonstructural proteins (31). The structural proteins are the core or capsid protein (C); a premembrane protein (prM), which is cleaved to yield the membrane protein in the mature virion; and the envelope protein (E). The latter two are glycosylated. The E protein shares significant homology with the E proteins of other flaviviruses, particularly those of the other members of the Japanese encephalitis virus (JEV) serocomplex, JEV itself, St. Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus. Antibodies directed against particular epitopes contained within the E protein are capable of virus neutralization. These epitopes have recently been mapped to at least two of three domains of the E protein, domains II and III, using sets of monoclonal antibodies for dengue virus (DENV) (6), as well as JEV (20) and WNV (1, 41). Neutralizing antibodies reacting with domain III are generally specific for each virus and do not cross-neutralize other viruses (or other serotypes of the same virus if multiple serotypes exist), while those targeting domain II are often cross-reactive. A high titer of virus-neutralizing antibodies is generally accepted as the best in vitro correlate of in vivo protection against virus infection or immunity to subsequent infection (23, 45). For this reason, the E protein was selected as the appropriate immunogen for use in the development of a WNV vaccine candidate.In previous studies at Hawaii Biotech, Inc. (HBI), a proprietary method of expression was used successfully to produce recombinant E proteins from flaviviruses, such as DENV serotypes 1 to 4, JEV, hepatitis C virus, and WNV (7, 19, 25, 26, 35). These proteins are truncated at the C terminus, leaving 80% of the native E protein (80E). The truncation deletes the membrane anchor portion of the protein, thus allowing it to be secreted into the extracellular medium, facilitating recovery. Furthermore, the expressed DENV and WNV proteins have been shown to be properly glycosylated and to maintain native conformation as determined by reactivity with conformationally sensitive monoclonal antibodies 4G2 and 9D12 (B. -A. Coller, D. E. Clements, and G. S. Bignami, unpublished data) and X-ray crystallography structure determination (25, 26). The immunogenicity of the vaccine formulations using the truncated WNV E protein (WN-80E [amino acids 1 to 401]) was demonstrated in mice (19), and its protective efficacy documented in hamsters (39, 43) and geese (15). The present report for the first time documents the immunogenicity and protective efficacy of a WN-80E vaccine formulation in a nonhuman primate animal model.     

产肠毒素大肠杆菌混合载体疫苗的免疫原性评价

本研究在构建表达ETEC菌毛抗原CFA/I、CS6和CS3、融合肠毒素LTB/STm的混合活载体疫苗的基础上 ,以两株福氏志贺载体疫苗 ,同等剂量相混合进行免疫。通过口服免疫 ,该混合多价活疫苗能够诱导机体产生相应的抗ETEC特异的血清和黏膜抗原抗体反应 ,达到了与各单独疫苗株一致的免疫效果 ,同时保持了载体志贺菌自身的免疫原性。     

Evaluation on Immunogenicity and Immune Efficacy of Different Kinds of Hepatitis B Vaccines

Hepatitis B vaccine, as the first high-effective recombinant commercial vaccine, was successfully developed inthe early 1980s. Since then, different opinions have occurred on the quality of vaccines with rapid developmentof target gene selecting, antigen expression system, andquality evaluation. Different antigens of hepatitis B vaccines are derived from different expression system, andthere are also some differences on manufacture procedureor glycosylated degree of antigen.     

Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

Background:Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infections and the resulting disease,coronavirus disease 2019(Covid-19),have spread to millions of persons worldwide.Multiple vaccine candidates are under development,but no vaccine is currently available.Interim safety and immunogenicity data about the vaccine candidate BNT162b1 in younger adults have been reported previously from trials in Germany and the United States.     

脂质体佐剂对增强HBsAg免疫原性的作用

利用DC Chol制备粒径为 5 0～ 30 0nm的正电荷脂质体 ,作为乙肝疫苗 (HBsAg)的佐剂 ,免疫BALB/c小鼠后进行血清中特异性抗体IgG1及IgG2a、脾细胞产生细胞因子的检测。结果该脂质体佐剂所诱导的抗体亚类以IgG2a为主 ,脾细胞产生的IL 2、IL 5、IFN γ分别比铝佐剂组高 16 5倍、 10倍、 2倍。表明该脂质体佐剂可以诱导很强的细胞免疫反应 ,是一种能促进Th1和Th2均衡应答的佐剂 ,值得作进一步的研究     

Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen

The spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a “genetically deamidated” form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity.