Spatio-temporal spread and evolution of Lassa virus in West Africa.

BACKGROUND: Lassa fever is a hemorrhagic disease caused by Lassa virus (LASV), which has been classified by the World Health Organization as one of the top infectious diseases requiring prioritized research. Previous studies have provided insights into the classification and geographic characteristics of LASV lineages. However, the factor of the distribution and evolution characteristics and phylodynamics of the virus was still limited. METHODS: To enhance comprehensive understanding of LASV, we employed phylogenetic analysis, reassortment and recombination detection, and variation evaluation utilizing publicly available viral genome sequences. RESULTS: The results showed the estimated the root of time of the most recent common ancestor (TMRCA) for large (L) segment was approximately 634 (95% HPD: [385879]), whereas the TMRCA for small (S) segment was around 1224 (95% HPD: [10301401]). LASV primarily spread from east to west in West Africa through two routes, and in route 2, the virus independently spread to surrounding countries through Liberia, resulting in a wider spread of LASV. From 1969 to 2018, the effective population size experienced two significant increased, indicating the enhanced genetic diversity of LASV. We also found the evolution rate of L segment was faster than S segment, further results showed zinc-binding protein had the fastest evolution rate. Reassortment events were detected in multiple lineages including sub-lineage IIg, while recombination events were observed within lineage V. Significant amino acid changes in the glycoprotein precursor of LASV were identified, demonstrating sequence diversity among lineages in LASV. CONCLUSION: This study comprehensively elucidated the transmission and evolution of LASV in West Africa, providing detailed insights into reassortment events, recombination events, and amino acid variations.

A novel Galleria mellonella experimental model for zoonotic pathogen Brucella.

Brucellosis is a major threat to public health and animal husbandry. Several in vivo vertebrate models, such as mice, guinea pigs, and nonhuman primates, have been used to study Brucella pathogenesis, bacteria-host interactions, and vaccine efficacy. However, these models have limitations whereas the invertebrate Galleria mellonella model is a cost-effective and ethical alternative. The aim of the present study was to examine the invertebrate G. mellonella as an in vivo infection model for Brucella. Infection assays were employed to validate the fitness of the larval model for Brucella infection and virulence evaluation. The protective efficacy of immune sera was evaluated by pre-incubated with a lethal dose of bacteria before infection. The consistency between the mouse model and the larval model was confirmed by assessing the protective efficacy of two Brucella vaccine strains. The results show that G. mellonella could be infected by Brucella strains, in a dose- and temperature-dependent way. Moreover, this larval model can effectively evaluate the virulence of Brucella strains in a manner consistent with that of mammalian infection models. Importantly, this model can assess the protective efficacy of vaccine immune sera within a day. Further investigation implied that haemolymph played a crucial role in the protective efficacy of immune sera. In conclusion, G. mellonella could serve as a quick, efficient, and reliable model for evaluating the virulence of Brucella strains and efficacy of immune sera in an ethical manner.

Nipah virus attachment glycoprotein ectodomain delivered by type 5 adenovirus vector elicits broad immune response against NiV and HeV.

Nipah virus (NiV) and Hendra virus (HeV) are newly emerging dangerous zoonotic pathogens of the Henipavirus genus of the Paramyxoviridae family. NiV and HeV (HNVs) which are transmitted by bats cause acute respiratory disease and fatal encephalitis in humans. To date, as there is a lack of antiviral drugs or effective antiviral therapies, the development of vaccines against those two viruses is of primary importance, and the immunogen design is crucial to the success of vaccines. In this study, the full-length protein (G), the ectodomain (Ge) and the head domain (Gs) of NiV attachment glycoprotein were delivered by the replication-defective type 5 adenovirus vector (Ad5) respectively, and the recombinant Ad5-NiV vaccine candidates (Ad5-NiVG, Ad5-NiVGe and Ad5-NiVGs) were constructed and their immunogenicity were evaluated in mice. The results showed that all the vaccine candidates stimulated specific humoral and cellular immune responses efficiently and rapidly against both NiV and HeV, and the Ad5-NiVGe elicited the strongest immune responses after a single-dose immunization. Furthermore, the potent conserved T-cell epitope DTLYFPAVGFL shared by NiV and HeV was identified in the study, which may provide valid information on the mechanism of HNVs-specific cellular immunity. In summary, this study demonstrates that the Ad5-NiVGe could be a potent vaccine candidate against HNVs by inducing robust humoral and cellular immune responses.

Comparative evaluation of protective immunity against Francisella tularensis induced by subunit or adenovirus-vectored vaccines.

Tularemia is a highly contagious disease caused by infection with Francisella tularensis (Ft), a pathogenic intracellular gram-negative bacterium that infects a wide range of animals and causes severe disease and death in people, making it a public health concern. Vaccines are the most effective way to prevent tularemia. However, there are no Food and Drug Administration (FDA)-approved Ft vaccines thus far due to safety concerns. Herein, three membrane proteins of Ft, Tul4, OmpA, and FopA, and a molecular chaperone, DnaK, were identified as potential protective antigens using a multifactor protective antigen platform. Moreover, the recombinant DnaK, FopA, and Tul4 protein vaccines elicited a high level of IgG antibodies but did not protect against challenge. In contrast, protective immunity was elicited by a replication-defective human type 5 adenovirus (Ad5) encoding the Tul4, OmpA, FopA, and DnaK proteins (Ad5-Tul4, Ad5-OmpA, Ad5-FopA, and Ad5-DnaK) after a single immunization, and all Ad5-based vaccines stimulated a Th1-biased immune response. Moreover, intramuscular and intranasal vaccination with Ad5-Tul4 using the prime-boost strategy effectively eliminated Ft lung, spleen and liver colonization and provided nearly 80% protection against intranasal challenge with the Ft live vaccine strain (LVS). Only intramuscular, not intranasal vaccination, with Ad5-Tul4 protected mice from intraperitoneal challenge. This study provides a comprehensive comparison of protective immunity against Ft provided by subunit or adenovirus-vectored vaccines and suggests that mucosal vaccination with Ad5-Tul4 may yield desirable protective efficacy against mucosal infection, while intramuscular vaccination offers greater overall protection against intraperitoneal tularemia.

Precise modification of the surface charge of antigen enhances vaccine immunogenicity.

Aluminum (alum) adjuvant is the most extensively used protein subunit vaccine adjuvant, and its effectiveness and safety have been widely recognized. The surface charge of the antigen determines its electrostatic adsorption to alum adjuvant, which directly affects the immune efficacy of the protein vaccine. In our study, we precisely modified its surface charge by inserting charged amino acids into the flexible region of the SARS-CoV-2 receptor-binding domain (RBD), achieving electrostatic adsorption and a site-specific anchor between the immunogen and alum adjuvant. This innovative strategy extended the bioavailability of the RBD and directionally displayed the neutralizing epitopes, thereby significantly enhancing humoral and cellular immunity. Furthermore, the required dose of antigen and alum adjuvant was greatly reduced, which improved the safety and accessibility of the protein subunit vaccine. On this basis, the wide applicability of this novel strategy to a series of representative pathogen antigens such as SARS-RBD, MERS-RBD, Mpox-M1, MenB-fHbp, and Tularemia-Tul4 was further confirmed. Charge modification of antigens provides a straightforward approach for antigenicity optimization of alum-adjuvanted vaccines, which has great potential to be adopted as a global defense against infectious diseases.

Challenges and developments in universal vaccine design against SARS-CoV-2 variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had become a global concern because of its unexpectedly high pathogenicity and transmissibility. SARS-CoV-2 variants that reduce the immune protection elicited from previous vaccination or natural infection raise challenges in controlling the spread of the pandemic. The development of universal vaccines against these variants seems to be a practical solution to alleviate the physical and economic effects caused by this disease, but it is hard to achieve. In this review, we describe the high mutation rate of RNA viruses and dynamic molecular structures of SARS-CoV-2 variants in several major neutralizing epitopes, trying to answer the question of why universal vaccines are difficult to design. Understanding the biological basis of immune evasion is crucial for combating these obstacles. We then summarize several advancements worthy of further study, including heterologous prime-boost regimens, construction of chimeric immunogens, design of protein nanoparticle antigens, and utilization of conserved neutralizing epitopes. The fact that some immunogens can induce cross-reactive immune responses against heterologous coronaviruses provides hints for universal vaccine development. We hope this review can provide inspiration to current universal vaccine studies.

A novel built-in adjuvant metallothionein-3 aids protein antigens to induce rapid, robust, and durable immune responses.

Adjuvants are crucial components of vaccines that can enhance and modulate antigen-specific immune responses. Herein, we reported for the first time that human metallothionein-3 (MT3), a low molecular weight cysteine-rich metal-binding protein, was a novel promising adjuvant candidate that could help protein antigens to induce rapid, effective, and durable antigen-specific immune responses. In the present study, MT3 was fused to outer membrane protein 19 (Omp19) of Brucella abortus (MT3-Omp19, MO) and C fragment heavy chain (Hc) of tetanus neurotoxin (MT3-Hc, MH), respectively. The results showed that MT3 as a built-in adjuvant increased the Omp19- or Hc-specific antibody responses by 100-1000 folds in seven days after primary immunization. Compared to other commercially available adjuvants, MT3 could stimulate earlier (4 days after primary injection) and stronger (10-100 folds) antibody response with lower antigen dose, and its adjuvanticity relied on fusion to antigen. Although the mechanism was not clear yet, the fusion protein MO was observed to directly activate DCs, promote germinal center formation and improve the speed of Ig class switching. Interestingly, our subsequent study found that other members of the mammalian MT family (human MT1 or murine MT3 for examples) also had potential adjuvant effects, but their effects were lower than human MT3. Overall, this study explored a new function of human MT3 as a novel built-in adjuvant, which may have important clinical application potential in vaccine development against global pandemics.

Comparative characterization of antibody responses induced by Ad5-vectored spike proteins of emerging SARS-CoV-2 VOCs.

Highly divergent SARS-CoV-2 variants have continuously emerged and spread around the world, and updated vaccines and innovative vaccination strategies are urgently needed to address the global SARS-COV2 pandemic. Here, we established a series of Ad5-vectored SARS-CoV-2 variant vaccines encoding multiple spike proteins derived from the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron lineages and analyzed the antibody immune responses induced by single-dose and prime-boost vaccination strategies against emerging SARS-CoV-2 variants of concern (VOCs). Single-dose vaccination with SARS-CoV-2 variant vaccines tended to elicit the optimal self-matched neutralizing effects, and Ad5-B.1.351 produced more broad-spectrum cross-neutralizing antibodies against diverse variants. In contrast, prime-boost vaccination further strengthened and broadened the neutralizing antibody responses against highly divergent SARS-CoV-2 variants. The heterologous administration of Ad5-B.1.617.2 and Ad5-B.1.429 to Ad5-WT-primed mice resulted in superior antibody responses against most VOCs. In particular, the Omicron spike could only stimulate self-matched neutralizing antibodies with infrequent cross-reactivities to other variants used in single-dose vaccination strategies; moreover, with prime-boost regimens, this vaccine elicited an optimal specific neutralizing antibody response to Omicron, and prompted cross-antibody responses against other VOCs that were very similar to those obtained with Ad5-WT booster. Overall, this study delineated the unique characteristics of antibody responses to the SARS-CoV-2 VOC spikes with the single-dose or prime-boost vaccination strategies and provided insight into the vaccine development of next SARS-CoV-2 VOCs.

Broadly neutralizing antibodies against Omicron-included SARS-CoV-2 variants induced by vaccination.

The SARS-CoV-2 Omicron variant shows substantial resistance to neutralization by infection- and vaccination-induced antibodies, highlighting the demands for research on the continuing discovery of broadly neutralizing antibodies (bnAbs). Here, we developed a panel of bnAbs against Omicron and other variants of concern (VOCs) elicited by vaccination of adenovirus-vectored COVID-19 vaccine (Ad5-nCoV). We also investigated the human longitudinal antibody responses following vaccination and demonstrated how the bnAbs evolved over time. A monoclonal antibody (mAb), named ZWD12, exhibited potent and broad neutralization against SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa, Delta, and Omicron by blocking the spike protein binding to the angiotensin-converting enzyme 2 (ACE2) and provided complete protection in the challenged prophylactic and therapeutic K18-hACE2 transgenic mouse model. We defined the ZWD12 epitope by determining its structure in complex with the spike (S) protein via cryo-electron microscopy. This study affords the potential to develop broadly therapeutic mAb drugs and suggests that the RBD epitope bound by ZWD12 is a rational target for the design of a broad spectrum of vaccines.

Screening of potential vaccine candidates against pathogenic Brucella spp. using compositive reverse vaccinology.

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and various animals. The threat of brucellosis has increased, yet currently available live attenuated vaccines still have drawbacks. Therefore, subunit vaccines, produced using protein antigens and having the advantage of being safe, cost-effective and efficacious, are urgently needed. In this study, we used core proteome analysis and a compositive RV methodology to screen potential broad-spectrum antigens against 213 pathogenic strains of Brucella spp. with worldwide geographic distribution. Candidate proteins were scored according to six biological features: subcellular localization, antigen similarity, antigenicity, mature epitope density, virulence, and adhesion probability. In the RV analysis, a total 32 candidate antigens were picked out. Of these, three proteins were selected for assessment of immunogenicity and preliminary protection in a mouse model: outer membrane protein Omp19 (used as a positive control), type IV secretion system (T4SS) protein VirB8, and type I secretion system (T1SS) protein HlyD. These three antigens with a high degree of conservation could induce specific humoral and cellular immune responses. Omp19, VirB8 and HlyD could substantially reduce the organ bacterial load of B. abortus S19 in mice and provide varying degrees of protection. In this study, we demonstrated the effectiveness of this unique strategy for the screening of potential broad-spectrum antigens against Brucella. Further evaluation is needed to identify the levels of protection conferred by the vaccine antigens against wild-type pathogenic Brucella species challenge.

Fc-Based Recombinant Henipavirus Vaccines Elicit Broad Neutralizing Antibody Responses in Mice.

The genus Henipavirus (HNVs) includes two fatal viruses, namely Nipah virus (NiV) and Hendra virus (HeV). Since 1994, NiV and HeV have been endemic to the Asia-Pacific region and responsible for more than 600 cases of infections. Two emerging HNVs, Ghana virus (GhV) and Mojiang virus (MojV), are speculated to be associated with unrecognized human diseases in Africa and China, respectively. Despite many efforts to develop vaccines against henipaviral diseases, there is presently no licensed human vaccine. As HNVs are highly pathogenic and diverse, it is necessary to develop universal vaccines to prevent future outbreaks. The attachment enveloped glycoprotein (G protein) of HNVs mediates HNV attachment to the host cell's surface receptors. G proteins have been used as a protective antigen in many vaccine candidates for HNVs. We performed quantitative studies on the antibody responses elicited by the G proteins of NiV, HeV, GhV, and MojV. We found that the G proteins of NiV and HeV elicited only a limited cross-reactive antibody response. Further, there was no cross-protection between MojV, GhV, and highly pathogenic HNVs. We then constructed a bivalent vaccine where the G proteins of NiV and HeV were fused with the human IgG1 Fc domain. The immunogenicity of the bivalent vaccine was compared with that of monovalent vaccines. Our results revealed that the Fc-based bivalent vaccine elicited a potent antibody response against both NiV and HeV. We also constructed a tetravalent Fc heterodimer fusion protein that contains the G protein domains of four HNVs. Immunization with the tetravalent vaccine elicited broad antibody responses against NiV, HeV, GhV, and MojV in mice, indicating compatibility among the four antigens in the Fc-fusion protein. These data suggest that our novel bivalent and tetravalent Fc-fusion proteins may be efficient candidates to prevent HNV infection.

Polysaccharide PCP-I isolated from Poria cocos enhances the immunogenicity and protection of an anthrax protective antigen-based vaccine.

Polysaccharides isolated from natural plants may represent a novel source of vaccine adjuvants. In this research, we focused on a natural plant polysaccharide, PCP-I, which is derived from Poria cocos, a Chinese traditional herbal medicine. We chose the anthrax protective antigen (PA) as a model to evaluate the adjuvant ability of PCP-I in enhancing the immunogenicity and protection of a PA-based anthrax vaccine. According to our results, PCP-I could significantly enhance anthrax specific anti-PA antibodies, toxin-neutralizing antibodies, anti-PA antibody affinity, as well as IgG1 and IgG2a levels. Besides, PCP-I increased the frequency of PA-specific memory B cells, increased the proliferation of PA-specific splenocytes, significantly stimulated the secretion of IL-4, and enhanced the activation of Dendritic cells (DCs) in vitro. The combination of PCP-I and CpG significantly enhanced the level of anti-PA antibodies and neutralizing antibodies, particularly PA-specific IgG2a, and shifted the Th2-bias to a Th1/Th2 balanced response. In addition, PCP-I with or without CpG could significantly improve the survival rate of immunized mice following challenge with the anthrax lethal toxin. These findings suggest that PCP-I may be a promising vaccine adjuvant that warrants further study.

Self-conjugated protective antigen elicits strong and durable protective antibody response against anthrax.

Anthrax is an acute and highly lethal disease caused by Bacillus anthracis. Protective antigen (PA) is the primary candidate antigen for the anthrax vaccines. However, PA suffers from poor immunogenicity with short-term anti-PA antibody response. High effectiveness, durable immunity, and minimal risk are required for development of an effective anthrax vaccine. In the present study, PA was self-conjugated by 8-arm polyethylene glycol (PEG) and further by thioester chemistry. As a result, 3-5 PA molecules were covalently conjugated and functioned as an antigen delivery system. The conjugate (PA-PEG) could maintain the structural properties of PA and increase the thermal stability of PA. PA-PEG could elicit a robust anti-PA IgG and neutralization antibody response in the magnitude and quality. The antibodies could be largely maintained for 180 days after three immunizations of PA-PEG. PA-PEG effectively stimulated the maturation of dendritic cell and rapidly induced the germinal center (GC) reaction. The percentages of the GC B-cells and T follicular helper (Tfh) cells were thus significantly augmented. The inflammatory response elicited by PA-PEG was comparable to those by PBS and PA. Therefore, PA-PEG is expected as an effective anthrax vaccine candidate with durable immunoprotection against anthrax.

Targeting the R domain of coagulase by active vaccination protects mice against lethal Staphylococcus aureus infection.

Coagulase (Coa) secreted by Staphylococcus aureus is associated with the establishment of staphylococcal disease, which activates host prothrombin and generates fibrin shields. The R domain of Coa, consisting of several conserved repeats, is important in immune evasion during S. aureus infection. However, previous research showed that the Coa R domain induced very weak specific antibody responses. In this study, we constructed a new R domain, CoaR6, consisting of 6 repeats that occur most frequently in clinical isolates. By fusing CoaR6 with Hc, the C-terminal fragment of the heavy chain of tetanus neurotoxin, we successfully increased anti-CoaR6 IgG levels in immunized mice which were hardly detected in mice immunized with CoaR6 plus alum. To further improve anti-CoaR6 responses, the combination adjuvants alum plus CpG were formulated with the antigen and exhibited a significantly higher specific antibody response. Moreover, active Th1/Th17 immune responses were observed in Hc-CoaR6 immunized group rather than CoaR6. Active immunization of Hc-CoaR6 with alum plus CpG showed protective effects in a peritonitis model induced by two S. aureus strains with different coagulase types. Our results provided strategies to improve the immunogenicity of R domain and supporting evidences for R domain to be an S. aureus vaccine candidate.

Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality.

Adenoviral vectors (AdV) are considered promising candidates for vaccine applications. A prominent group of Toll-like receptors (TLRs) participate in the adenovirus-induced adaptive immune response, yet there is little information regarding the role of TLR4 in AdV-induced immune responses in recent literature. We investigated the function of TLR4 in both adaptive and innate immune responses to an AdV-based anthrax vaccine. By immunizing wild-type and TLR4 knockout (TLR4-KO) mice, we revealed the requirement of TLR4 in AdV-induced innate responses. We also showed that TLR4 functions are required for germinal centre responses in immunized mice, as expression of the apoptosis-related marker Fas was down-regulated on germinal centre B cells from TLR4-KO mice. Likewise, decreased expression of inducible costimulator on follicular T helper cells was observed in immunized TLR4-KO mice. Moreover, a potent protective antigen-specific humoral immune response was mimicked using an adjuvant system containing the TLR4 agonist monophosphoryl lipid A. Overall, our findings showed that very rapid antigen-specific antibody production is correlated with the TLR4-imprinted germinal centre response to AdV-based vaccine. These results provide additional evidence for the use of the AdV and a TLR agonist to induce humoral responses. Our findings offer new insights into rational vaccine design.

Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses.

Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic balance under stress. In conclusion, our results provide a better understanding of the global metabolic adaptations of B. abortus associated with distinct environmental stresses. The identification of proteins necessary for stress resistance is crucial toward elucidating the infectious process in order to control brucellosis, and may facilitate the discovery of novel therapeutic targets and effective vaccines.

A comprehensive proteogenomic study of the human Brucella vaccine strain 104 M.

BACKGROUND: Brucella spp. are Gram-negative, facultative intracellular pathogens that cause brucellosis in both humans and animals. The B. abortus vaccine strain 104 M is the only vaccine available in China for the prevention of brucellosis in humans. Although the B. abortus 104 M genome has been fully sequenced, the current genome annotations are not yet complete. In addition, the main mechanisms underpinning its residual toxicity and vaccine-induced immune protection have yet to be elucidated. Mapping the proteome of B. abortus 104 M will help to improve genome annotation quality, thereby facilitating a greater understanding of its biology. RESULTS: In this study, we utilized a proteogenomic approach that combined subcellular fractionation and peptide fractionation to perform a whole-proteome analysis and genome reannotation of B. abortus 104 M using high-resolution mass spectrometry. In total, 1,729 proteins (56.3% of 3,072) including 218 hypothetical proteins were identified using the culture conditions that were employed this study. The annotations of the B. abortus 104 M genome were also refined following identification and validation by reverse transcription-PCR. In addition, 14 pivotal virulence factors and 17 known protective antigens known to be involved in residual toxicity and immune protection were confirmed at the protein level following induction by the 104 M vaccine. Moreover, a further insight into the cell biology of multichromosomal bacteria was obtained following the elucidation of differences in protein expression levels between the small and large chromosomes. CONCLUSIONS: The work presented in this report used a proteogenomic approach to perform whole-proteome analysis and genome reannotation in B. abortus 104 M; this work helped to improve genome annotation quality. Our analysis of virulence factors, protective antigens and other protein effectors provided the basis for further research to elucidate the mechanisms of residual toxicity and immune protection induced by the 104 M vaccine. Finally, the potential link between replication dynamics, gene function, and protein expression levels in this multichromosomal bacterium was detailed.

Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R.

Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2-) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R.