Immunogenicity and immunoprotection of the functional TL-HN fragment derived from tetanus toxin.

Tetanus toxin (TeNT) is a protein toxin produced by Clostridium tetani bacteria, which causes hyperreflexia and rhabdomyolysis by spastic paralysis. Like botulinum neurotoxin, TeNT comprises a heavy chain (HC) and a light chain (LC) linked via an interchain disulfide bond, which include the following three functional domains: a receptor-binding domain (Hc), a translocation domain (HN), and a catalytic domain (LC). Herein, we produced and characterized three functional domains of TeNT and three types of TeNT-derived L-HN fragments (TL-HN, TL-GS-HN and TL-2A-HN), which contained L and HN domains but lacked the Hc domain. The immunological effects of these different functional domains or fragments of TeNT were explored in an animal model. Our investigations showed the TL-HN functional fragment provided the best immunoprotection among all the TeNT functional domains. The TL-HN fragment, as a protective antigen, induced the highest levels of neutralizing antibodies, indicating that it might contain some crucial epitopes. Further experiments revealed that the protective effect of TL-HN was superior to that of the THc, TL, or THN fragments, either individually or in combination. Therefore, the TL-HN fragment exerts an important function in immune protection against tetanus toxin, providing a good basis for the development of TeNT vaccines or antibodies, and could serve as a promising subunit vaccine to replace THc or tetanus toxoid (TT).

Tetanus toxin and botulinum neurotoxin-derived fusion molecules are effective bivalent vaccines.

Tetanus toxin (TeNT) and botulinum neurotoxins (BoNTs) are neuroprotein toxins, with the latter being the most toxic known protein. They are structurally similar and contain three functional domains: an N-terminal catalytic domain (light chain), an internal heavy-chain translocation domain (HN domain), and a C-terminal heavy chain receptor binding domain (Hc domain or RBD). In this study, fusion functional domain molecules consisting of the TeNT RBD (THc) and the BoNT/A RBD (AHc) (i.e., THc-Linker-AHc and AHc-Linker-THc) were designed, prepared, and identified. The interaction of each Hc domain and the ganglioside receptor (GT1b) or the receptor synaptic vesicle glycoprotein 2 (SV2) was explored in vitro. Their immune response characteristics and protective efficacy were investigated in animal models. The recombinant THc-linker-AHc and AHc-linker-THc proteins with the binding activity had the correct size and structure, thus representing novel subunit vaccines. THc-linker-AHc and AHc-linker-THc induced high levels of specific neutralizing antibodies, and showed strong immune protective efficacy against both toxins. The high antibody titers against the two novel fusion domain molecules and against individual THc and AHc suggested that the THc and AHc domains, as antigens in the fusion functional domain molecules, do not interact with each other and retain their full key epitopes responsible for inducing neutralizing antibodies. Thus, the recombinant THc-linker-AHc and AHc-linker-THc molecules are strong and effective bivalent biotoxin vaccines, protecting against two biotoxins simultaneously. Our experimental design will be valuable to develop recombinant double-RBD fusion molecules as potent bivalent subunit vaccines against bio-toxins. KEY POINTS: • Double-RBD fusion molecules from two toxins had the correct structure and activity. • THc-linker-AHc and AHc-linker-THc efficiently protected against both biotoxins. • Such bivalent biotoxin vaccines based on the RBD are a valuable experimental design.

Biology activity and characterization of the functional L-HN fragment derivative of botulinum neurotoxin serotype E.

OBJECTIVES: The mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. METHODS: Neurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. RESULTS: We found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 µg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. CONCLUSIONS: The EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.

Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs.

Botulinum neurotoxin (BoNTs; serotypes A, B, E, and F) cause botulism disease in humans, which could be effectively treated using antitoxins. Herein, we established a novel receptor-binding domain (RBD)-based antitoxin using recombinant C terminal heavy chain (Hc) domains of BoNTs as immunogens. Immunization of horses with these recombinant Hc domains allowed the purification and digestion of IgGs from hyper-immune sera to produce high-quality and high-efficiency monovalent botulism antitoxin F(ab')2 against each BoNT (M-BATs). However, these M-BATs could not bind or neutralize other serotypes of BoNTs, and that there were no cross-protective effects among these M-BATs. This suggested the need to prepare tetravalent antitoxins to neutralize the four BoNTs simultaneously. Thus, these M-BATs were formulated into a novel tetravalent botulism antitoxin (T-BAT), in which a 10-ml volume contained 10000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins. The novel antitoxin preparation could prevent and treat the four mixed botulinum neurotoxins simultaneously in vivo, representing strong efficacy in an animal poisoning model. Moreover, these antibodies in T-BAT could bind the RBD, whereas conventional antitoxins based on inactivated toxins mainly bind the light chain or heavy chain translocation domain (HN) and weakly bind the important RBD in current experimental conditions. The high levels of RBD-specific novel antitoxins can efficiently bind the RBD and neutralize natural or recombinant toxins containing this RBD. The findings of the present study experimentally support the use of RBD-specific antitoxins to treat BoNT serotype A, B, E, and F-mediated botulism. This study demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of these toxins as an alternative antigen to inactivated toxins. KEY POINTS: • Antitoxins based on the receptor-binding domains of botulinum neurotoxins were made. • Novel antitoxin binds RBD; traditional antitoxin mainly binds light chain or HN domain. • A tetravalent antitoxin could prevent and treat the four mixed neurotoxins in vivo.

Development and evaluation of a tetravalent botulinum vaccine.

Botulinum neurotoxins (BoNTs) are the most toxic known proteins. Naturally occurring botulism in humans is caused by botulinum serotypes A, B, E, and F. Vaccination is an effective strategy to prevent botulism. In this study, a tetravalent botulinum vaccine (TBV) that can prevent serotypes A, B, E, and F was developed using the C-terminal receptor-binding domain of BoNT (Hc) as an antigen. To develop a suitable vaccine formulation, in vitro binding experiments of antigens and aluminum adjuvant in different buffers, and in vivo experiments of TBV at different antigen concentrations, were conducted. Our results showed that the optimal vaccine formulation buffer was a pH 6.0 phosphate buffer, and the suitable antigen concentration was 40 or 80 µg/ml of each antigen. A pilot-scale TBV was then prepared and evaluated for immunogenicity and stability. The results showed that TBV could elicit strong protective efficacy against each BoNT in mice, and remain effective after two years of storage at 4ºC, indicating that the preparation was stable and highly effective. Adsorption experiments also showed that the antigens could be well adsorbed by the aluminum adjuvant after 2 years of storage. Our results provide valuable experimental data supporting the development of a tetravalent botulinum vaccine, which is a promising candidate for the prevention of botulinum serotypes A, B, E, and F.

Recombinant L-HN Fusion Antigen Derived from the L and HN Domains of Botulinum Neurotoxin B Stimulates a Protective Antibody Response Against Active Neurotoxin.

Botulinum neurotoxin (BoNT) is a neurotoxin produced by Clostridium botulinum in an anaerobic environment. BoNT is the most toxic protein among bacteria, animals, plants, and chemical substances reported to date. BoNTs are 150 kDa proteins composed of three major functional domains: catalytic (L domain, 50 kDa), translocation (HN domain, 50 kDa), and receptor-binding (Hc domain, 50 kDa) domains. Most studies have focused on the use of the Hc domain as an antigen because it is capable of generating robust protective immunity and contains some functional neutralizing epitopes. In the present study, we produced and characterized a recombinant L-HN fusion fragment of the parent BoNT/B (BL-HN) composed of L and HN domains with a deletion in the Hc domain (BHc). When the BL-HN protein was expressed in E. coli, it retained its stable structure and antigenicity. As a vaccine antigen, the recombinant BL-HN protein was found to induce sufficient protection against native BoNT/B in a mouse model. The BL-HN subunit vaccine could also induce a strong humoral immune response and generate sufficient neutralizing antibodies in immunized mice. Therefore, BL-HN may retain the native neurotoxin structure and critical epitopes responsible for inducing serum neutralizing antibodies. Studies of the dose-dependent immunoprotective effects further confirmed that the BL-HN antigen could provide potent protective immunity. This finding suggests that BL-HN can play an important role in immune protection against BoNT/B. Therefore, the BL-HN fusion fragment provides an excellent platform for the design of recombinant botulinum vaccines and neutralizing antibodies.

Evaluation of a recombinant tetanus toxin subunit vaccine.

Tetanus is an acute, fatal disease caused by exotoxin produced by Clostridium tetani. The current vaccine against tetanus is based on inactivated tetanus toxin (TeNT). To develop a recombinant TeNT vaccine suitable for replacement of full-length tetanus toxoid (TT) vaccine for use in humans, a recombinant non-tagged isoform of the Hc domain of the tetanus toxin (THc) was expressed in Escherichia coli and purified by sequential chromatography steps. The immunogenicity and protective effect of the THc antigen were explored and compared with those of TT in Balb/c mice. The THc-based subunit vaccine provided complete protection against TeNT challenge following a high dosage as a toxoid vaccine. While the anti-THc and neutralising antibody titres were higher for the THc-based vaccine than the TT vaccine because protective epitopes are located on the THc domain. Frequency- and dose-dependent immunoprotection were also observed in THc-immunised mice. Mice immunised with one injection of 1 µg or 4 µg THc antigen were completely protected against 102 or 103 50% mouse lethal dose (LD50) of TeNT, respectively. Furthermore, the THc protein was found to recognise and bind to ganglioside GT1b in a dose-dependent manner, and anti-THc sera antibodies also inhibited binding between THc and GT1b. Antigen on the form of recombinant non-tagged THc domain expressed in E. coli achieved strong immunoprotective potency, suggesting that it could be developed into a candidate subunit vaccine against tetanus as an alternative to the current TT vaccine.

Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are highly toxic proteins that mediate their effects by binding to neuronal receptors and block the neutralizing ability of therapeutic antibodies. Vaccination is currently the most effective strategy to prevent botulism. In this study, a series of recombinant functional domain antigens of BoNT/A were prepared and identified, and their immunoprotective efficacies were explored and compared. Our results showed that all antigens produced strong humoral immune responses, although their protective effects against the toxin were different. Only the Hc and HN-L antigens produced strong protective effects and afforded complete immunoprotection. In addition, the combined vaccine groups showed that there was no synergistic effect on immune responses after antigen combination, suggesting that the integrity of the toxin antigen or domain is crucial to the immune effects. Studies of the dose-dependent immunoprotective effects further confirmed that the Hc domain antigen afforded more effective protective potency than the HN-L antigen, equivalent to the immune effect of the full-length toxin (Hc + HN-L combination group). Overall, our results demonstrated that the Hc domain elicited a strong protective immune response and also provided basic data and theoretical support for the development of Hc-based BoNT/A subunit vaccine. Therefore, the receptor binding domain Hc is implicated as a promising target antigen of the BoNT/A recombinant subunit vaccine as an alternative to the toxoid vaccine.

Development and evaluation of candidate subunit vaccine and novel antitoxin against botulinum neurotoxin serotype E.

Botulinum neurotoxins (BoNTs) are among the most toxic proteins. Vaccination is an effective strategy to prevent botulism. To generate a vaccine suitable for human use, a recombinant non-His-tagged isoform of the Hc domain of botulinum neurotoxin serotype E (rEHc) was expressed in Escherichia coli and purified by sequential chromatography. The immunogenicity of rEHc was evaluated in mice and dose- and time-dependent immune responses were observed in both antibody titers and protective potency. Then, the pilot-scale expression and purification of rEHc were performed, and its immunological activity was characterized. Our results showed rEHc has good immunogenicity and can elicit strong protective potency against botulinum neurotoxin serotype E (BoNT/E) in mice, indicating that rEHc is an effective botulism vaccine candidate. Further, we developed a novel antitoxin against BoNT/E by purifying F(ab')2 from pepsin-digested serum IgG of rEHc-inoculated horses. The protective effect of the F(ab')2 antitoxin was determined in vitro and in vivo. The results showed that our F(ab')2 antitoxin can prevent botulism in BoNT/E-challenged mice and effectively alleviate the progression of paralysis caused by BoNT/E to achieve therapeutic effects. Therefore, our results provide valuable experimental data for the production of a novel antitoxin, which is a promising candidate for the treatment of BoNT/E-induced botulism.

Development and evaluation of candidate subunit vaccine against botulinum neurotoxin serotype B.

Botulinum neurotoxins (BoNTs) are potential biological weapons because of their high toxicity and mortality. Vaccination is an effective strategy to prevent botulism. The carboxyl-terminus of the heavy chain (Hc domain) is nontoxic and sufficient to generate protective immune responses against natural BoNTs in animals. To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype B (BHc) was expressed in Escherichia coli and purified by sequential chromatography. The immunogenicity of recombinant E.coli-expressed BHc and the yeast-expressed mBHc antigens was explored and compared in Balb/c mice. BHc provided comparable protective potency but elicited significantly higher antibody titer and neutralization potency against BoNT/B after twice immunization, indicating that the recombinant BHc protein expressed in E.coli have better immunogenicity than the yeast-expressed mBHc. Moreover, a frequency and dose-dependent effect was observed in mice immunized with BHc subunit vaccine and the anti-BHc ELISA antibody titers correlated well with neutralizing antibody titers and protection potency. In summary, the Alhydrogel-formulated BHc subunit vaccine afforded effective protection against BoNT/B challenge. Therefore, the non-His-tagged and homogeneous BHc expressed in E.coli represents a good potential candidate subunit vaccine for human use.

A novel fully human anti-CD47 antibody as a potential therapy for human neoplasms with good safety.

Strategies for targeting CD47 are becoming a hot spot of cancer immunotherapy. However the ubiquitous expression of CD47, especially on the RBC, makes the targeted therapy facing safety risk issues. So, how to balance the safety and efficacy during CD47 inhibition is currently a major question. We had reported an anti-CD47 antibody ZF1 with potent anti-tumor effect. In this study, we further developed and assessed a novel fully human anti-CD47 antibody, AMMS4-G4, derived from ZF1 using affinity maturation. AMMS4-G4 exhibited equivalent anticancer effects with Hu5F9-G4, a humanized anti-CD47 antibody in clinical trial, on the potential of inducing significant phagocytosis of tumor cells in vitro and prolonging the survival of leukemia xenografted mice. Additionally, AMMS4-G4 significantly inhibited the growth of grafted solid tumors by enhancing macrophage infiltration and modestly enhanced the anti-tumor activity of opsonizing antibody and antiangiogenic therapy. In cynomolgus monkeys, AMMS4-G4 was safely administered, was well tolerated at doses of 30 and 60 mg/kg, and did not produce serious adverse events, except for the reversible anemia, which was observed after 3 days and started to recover from 9 days later. Remarkably, it was proved by in vitro assay that Hu5F9-G4 induced RBC hemagglutination which wasn't observed in AMMS4-G4. On the whole, AMMS4-G4 was demonstrated to be a promising candidate with great potential and safe profile for cancer immunotherapy.

Improved synaptic and cognitive function in aged 3 × Tg-AD mice with reduced amyloid-ß after immunotherapy with a novel recombinant 6Aß15-TF chimeric vaccine.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder impairing memory and cognition. In this study, we describe the immunogenicity and protective efficacy of the novel recombinant 6Aß15-TF chimeric antigen as a subunit protein vaccine for AD. Recombinant 6Aß15-TF chimeric vaccine induced strong Aß-specific humoral immune responses without Aß-specific T cell immunity in C57/BL6 and 3 × Tg-AD mice at different ages. As an early immunotherapy model for AD, this vaccine induced high titers of long-lasting anti-Aß42 antibodies in aged 3 × Tg-AD mice, which led to improve behavioral performance and markedly reduced the levels of insoluble and soluble Aß and Aß oligomers. In agreement with these findings, immunotherapy with 6Aß15-TF prevented the Aß-induced decrease of presynaptic and postsynaptic proteins in aged 3 × Tg-AD mice. Our results suggest that this novel and highly immunogenic recombinant 6Aß15-TF chimeric vaccine provides neuroprotection in AD mice and can be considered an effective AD candidate vaccine.

A Built-In CpG Adjuvant in RSV F Protein DNA Vaccine Drives a Th1 Polarized and Enhanced Protective Immune Response.

Human respiratory syncytial virus (RSV) is the most significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection.

Enhanced effects of DNA vaccine against botulinum neurotoxin serotype A by targeting antigen to dendritic cells.

As dendritic cells (DCs) play a critical role in priming antigen-specific immune responses, the efficacy of DNA vaccines may be enhanced by targeting the encoded antigen proteins to DCs. In this study, we constructed a DC-targeted DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A (AHc) fused with scDEC, a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Intramuscular injections of mice with the DC-targeted DNA vaccine (pVAX1-scDEC-AHc) stimulated more DCs to mature than the non-targeted DNA vaccine (pVAX1-SAHc) in the splenocytes. The DC-targeted DNA vaccine could induce more DCs maturation at the site of inoculation. The DC-targeted DNA vaccine induced stronger AHc-specific humoral immune responses, lymphocyte proliferative responses and protective potency against BoNT/A in mice than did pVAX1-SAHc. Moreover, the DC-targeting DNA vaccine provided effective protection after only two inoculations. In summary, these results showed that the DC-targeted fusion DNA vaccine could generate strong immunity, indicating that maturation of DCs induced by pVAX1-scDEC-AHc may be helpful for priming and boosting immune responses. Thus, we propose that the strategy of targeting antigen to DCs in vivo via DEC205 can enhance effectively the potency of DNA vaccines against BoNTs or other pathogens in an animal model.

Prophylactic immunotherapy of Alzheimer's disease using recombinant amyloid-ß B-cell epitope chimeric protein as subunit vaccine.

Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. The neuropathological features of the disease include senile plaques (SPs), neurofibrillary tangles (NFTs) and neuronal loss in affected brain regions. The amyloid cascade hypothesis suggests that production and accumulation of excessive amyloid-ß (Aß) may be the main cause in the onset and progression of Alzheimer's disease. Active and passive immunotherapy targeting Aß may be the most promising strategy to prevent or treat AD. This commentary focuses on the prophylactic immunotherapy of Alzheimer's disease using recombinant Aß B-cell epitope chimeric protein as subunit vaccine targeting amyloid-ß. We discuss the efficiency and perspective of this type of recombinant subunit protein vaccine and suggest a novel direction on the path to a successful AD immunotherapy. This novel chimeric protein immunogen as subunit vaccine of AD may be designed to mimic the assembly states of Aß42 or oligomers using multivalent foldable Aß1-15 (B cell epitopes of Aß42) and foreign T helper (Th) epitopes (as the T cell epitopes of Aß42) constructs.

Enhanced efficacy of DNA vaccination against botulinum neurotoxin serotype A by co-administration of plasmids encoding DC-stimulating Flt3L and MIP-3α cytokines.

Targeting antigens encoded by DNA vaccines to the key antigen-presenting cells by chemotactic or growth factors, is an effective strategy for enhancing the potency of DNA vaccinations. Here, we report the effects of chemotactic or growth factors on a DNA vaccine against botulinum neurotoxin serotype A (BoNT/A) in a mouse model. We demonstrated that mice immunized with DNA constructs encoding the Hc domain of BoNT/A (AHc) fused with DC-stimulating Flt3L or MIP-3α cytokines failed to elicit an enhanced or efficacious AHc-specific humoral or protective response in mice. However, the potency of DNA vaccination was significantly modulated and enhanced by co-administration of AHc-expressing DNA with pFlt3L or pMIP-3α, which generated strong immune and protective responses against BoNT/A. Moreover, the enhanced potency was further boosted by co-administration of AHc-expressing DNA with the combination of pFlt3L and pMIP-3α in mice, but not with the Flt3L-MIP-3α fusion molecule, which indicated that co-immunization with both pFlt3L and pMIP-3α could synergistically enhance AHc-specific immune and protective responses against BoNT/A. In summary, our findings indicate that co-administration of plasmids encoding antigen and cytokine rather than administration of plasmids encoding cytokine-antigen fusion is effective to enhance the potency of AHc-expressing DNA vaccine.

A novel recombinant 6Aß15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer's disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-ß (Aß) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aß15-THc-C immunogen was formulated with alum adjuvant as a novel Aß B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aß-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aß or oligomeric forms of Aß, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aß levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aß-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine.

A Novel Aß B-Cell Epitope Vaccine (rCV01) for Alzheimer's Disease Improved Synaptic and Cognitive Functions in 3 × Tg-AD Mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive amyloid-ß accumulation, loss of cognitive abilities, and synaptic alterations. Given the remarkable recovery of cognition in AD models of targeting-Aß immunotherapy, we sought to determine the molecular correlate(s) associated with improvement. We evaluated the efficacy of a recombinant chimeric 6Aß15-T antigen formulated with alum adjuvant as a novel Aß B-cell epitope vaccine (rCV01) in 3 × Tg-AD mice. rCV01 elicited robust Th2-polarized Aß-specific antibodies without autoimmune T cell responses in 3 × Tg-AD mice. The long-lasting anti-Aß42 antibodies were associated with markedly reduced AD-like pathology, enhanced synaptic function, and improved cognitive performance in aged 3 × Tg-AD mice. This is the first report to provide one hypothesis for the improved outcomes following vaccination is a reduction in the levels of active calpain in rCV01-immunized AD mice, which is likely attributable to preventing dynamin 1 and PSD-95 degradation allowing functional recovery of cognition. rCV01 is a highly immunogenic recombinant chimeric 6Aß15-T vaccine that shows clear neuroprotective properties in preclinical mouse models of AD and is a candidate for an effective AD vaccine.

Peripherally administered sera antibodies recognizing amyloid-ß oligomers mitigate Alzheimer's disease-like pathology and cognitive decline in aged 3× Tg-AD mice.

Active and passive immunotherapy targeting amyloid-ß (Aß) may be the most promising strategy to prevent or treat Alzheimer's disease (AD). Previously, immunization with the recombinant 6Aß15-T antigen generated robust anti-Aß serum antibodies that strongly recognized Aß42 oligomers in different mice, markedly reduced the amyloid burden, and improved behavioral performance of immunized older AD mice. Here, we further determined that these anti-6Aß15-T serum antibodies from different strains of mice displayed anti-Aß antibody responses against the same epitopes in the Aß1-15 region. Peripheral administration of anti-6Aß15-T serum antibodies was also effective to mitigate AD-like pathology and cognitive decline in aged 3× Tg-AD mice. Specifically, the levels of Aß and tau in the brains of 3× Tg-AD mice were significantly reduced after passive immunotherapy, which seemed necessary or beneficial to ameliorate memory impairment. In addition, our results showed that this immunotherapy also prevented presynaptic dynamin 1 degradation, which might help to further protect synaptic functions and allow functional recovery of cognition. Moreover, immunization with 6Aß15-T in rabbits induced a similar antibody response as that in mice, and the rabbit serum antibodies reacted strongly with Aß42 oligomers and inhibited oligomer-mediated neurotoxicity. We concluded that passive immunization with Aß42 oligomer conformation-sensitive anti-6Aß15-T serum antibodies is effective in providing potentially therapeutic effects in aged 3× Tg-AD mice by reducing Aß and tau.

Co-immunization with DNA and protein mixture: a safe and efficacious immunotherapeutic strategy for Alzheimer's disease in PDAPP mice.

Active immunotherapy targeting ß-amyloid (Aß) is the most promising strategy to prevent or treat Alzheimer's disease (AD). Based on pre-clinical studies and clinical trials, a safe and effective AD vaccine requires a delicate balance between providing therapeutically adequate anti-Aß antibodies and eliminating or suppressing unwanted adverse T cell-mediated inflammatory reactions. We describe here the immunological characterization and protective efficacy of co-immunization with a 6Aß15-T DNA and protein mixture without adjuvant as an AD immunotherapeutic strategy. Impressively, this co-immunization induced robust Th2-polarized Aß-specific antibodies while simultaneously suppressed unwanted inflammatory T cell reactions and avoiding Aß42-specific T cell-mediated autoimmune responses in immunized mice. Co-immunization with the DNA + protein vaccine could overcome Aß42-associated hypo-responsiveness and elicit long-term Aß-specific antibody responses, which helped to maintain antibody-mediated clearance of amyloid and accordingly alleviated AD symptoms in co-immunized PDAPP mice. Our DNA and protein combined vaccine, which could induce an anti-inflammatory Th2 immune response with high level Aß-specific antibodies and low level IFN-γ production, also demonstrated the capacity to inhibit amyloid accumulation and prevent cognitive dysfunction. Hence, co-immunization with antigen-matched DNA and protein may represent a novel and efficacious strategy for AD immunotherapy to eliminate T cell inflammatory reactions while retaining high level antibody responses.