Identification of Linear Peptide Immunogens with Verified Broad-spectrum Immunogenicity from the Conserved Regions within the Hemagglutinin Stem Domain of H1N1 Influenza Virus.

BACKGROUND: Influenza A viruses (IAVs) induce acute respiratory disease and cause severe epidemics and pandemics. Since IAVs exhibit antigenic variation and genome reassortment, the development of broad-spectrum influenza vaccines is crucial. The stem of the hemagglutinin (HA) is highly conserved across IAV strains and thus has been explored in broad-spectrum influenza vaccine studies. The present study aimed to identify viral epitopes capable of eliciting effective host immune responses, which can be explored for the development of broad-spectrum non-strain specific prophylactic options against IAV. METHODS: In this study, a series of conserved linear sequences from the HA stem of IAV (H1N1) was recognized by sequence alignment and B/T-cell epitope prediction after being chemically coupled to the Keyhole Limpet Hemocyanin (KLH) protein. The predicted linear epitopes were identified by enzyme-linked immunosorbent assay (ELISA) after animal immunization and then fused with ferritin carriers. RESULTS: Three predicted linear epitopes with relatively strong immunogenicity, P3, P6 and P8 were fused with ferritin carriers P3F, P6F and P8F, respectively to further improve their immunogenicity. Antibody titre of the sera of mice immunized with the recombinant immunogens revealed the elicitation of specific antibody-binding activities by the identified sequences. While hemagglutinin-inhibition activities were not detected in the antisera, neutralizing antibodies against the H1 and H3 virus subtypes were detected by the microneutralization assay. CONCLUSION: The linear epitopes fused with ferritin identified in this study can lay the foundation for future advancements in development of broad-spectrum subunit vaccine against IAV (H1N1), and give rise to the potential future applicability of ferritin-based antigen delivery nanoplatforms.

Self-assembling ferritin nanoparticles coupled with linear sequences from canine distemper virus haemagglutinin protein elicit robust immune responses.

BACKGROUND: Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. RESULT: The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. CONCLUSIONS: YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.

A novel small molecule displays two different binding modes during inhibiting H1N1 influenza A virus neuraminidases.

Neuraminidase (NA) inhibitors can suppress NA activity to block the release of progeny virions and are effective against influenza viruses. As potential anti-flu drugs with unique functions, NA inhibitors are greatly concerned by the worldwide scientists. It has been reported recently that one of the novel quindoline derivatives named 7a, could inhibit both A/Puerto Rico/8/34 (H1N1) NA (NAPR) and A/California/04/09 (H1N1) NA (NACA). However, potential structure differences in the active site could be easily detected between the NAPR and NACA according to the flexibilities of their 150-loops located catalytic site. And no obvious 150-cavity could be observed in NACA crystal structure. In order to explore whether 7a could trigger the inhibition against these two NAs in the same way, a serial molecular dynamics simulation approach were applied in this study. The results indicated that 7a could be adopted under a relatively extended pose in the active center of NAPR. While in NACA-7a complex, the derivate preferred to be recognized and located on the side of active center. Interestingly, the potential of 7a was also found to be able to change the flexibility of the 150-loop in NACA that is absent of 150-cavity. Furthermore, a 150-cavity-like architecture could be induced in the active site of NACA. The results of this study revealed two kinds of binding modes of this novel small molecule inhibitor against NAs that might provide a theoretical basis for proposing novel inhibition mechanism and developing future influenza A virus inhibitors.

Effects of poly(I:C) and MF59 co-adjuvants on immunogenicity and efficacy of survivin polypeptide immunogen against melanoma.

Malignant tumors pose a public health problem that jeopardizes human life and quality of living. At present, tumor vaccines in clinical research typically are aimed at stimulating the cellular immune response, while more effective vaccines should take into account the synergy between broad spectrum antibodies and high levels of cellular immunity. In this study, epitope peptides (68-81, 95-104, 80-88) of the tumor antigen survivin were chosen as immunogens and supplemented with poly(I:C) and/or MF59 adjuvant to evaluate the immune effects and anti-melanoma activities. The results indicated that poly(I:C) and MF59 could assist the survivin epitope peptide immunogen to control the tumor size, quality, and volume in black melanoma mouse models. Analyses by antibody titering, antibody isotyping and ELISPOT suggested that the adjuvanted immunogen could induce humoral immunity in mice. Poly(I:C) and MF59 combined with survivin peptide 95-104 could effectively induce humoral immunity mediated by type 2 T helper (Th2) cells. This study provides a basis for candidate immunogen design based on survivin and provides support for tumor therapy that can induce a more balanced Th1/Th2 immune response.

Effects of insulin on transcriptional response and permeability in an in vitro model of human blood-brain barrier.

Alzheimer's disease (AD) is the most prevalent form of dementia worldwide and is an emerging global epidemic. Active and passive immune therapies targeting beta amyloid (Aß) have shown very limited evidence in human studies of clinical benefits from these approaches. Epidemiological studies have shown that subjects with type 2 diabetes (T2D) are at higher risk of developing AD. However, whether and how these two conditions are causally linked is unknown. With the purpose of confirming the relationship between T2D and AD, this study specifically focused on effects of insulin in an in vitro model of the human blood-brain barrier (BBB) and on potential mechanisms of action in the treatment of AD. By using a series of assays to establish a BBB model, we demonstrated that insulin treatment alone could induce the increase of brain endothelial barrier properties. The transcriptional response of hCMEC/D3 cells to activation with different concentrations of insulin was determined by RT-PCR, and expression levels of genes involved in the control of barrier permeability, including inter-brain endothelial junctions, integrin-focal adhesions complexes, and transporter system, were found to be altered by the treatment. Notably, the influence of insulin on expression of the ATP-binding cassette (ABC) transporter which contributes to the clearance of Aß was investigated. Insulin up-regulated adherens junction and tight junction transmembrane proteins, as well as the ABC transporter. By treatment with insulin, the models have major advantages: it is fast, it has low cost, it is fit for considerable samples, and its conditions are under control.

A biepitope, adjuvant-free, self-assembled influenza nanovaccine provides cross-protection against H3N2 and H1N1 viruses in mice.

Currently, the incorporation of multiple epitopes into vaccines is more desirable than the incorporation of a single antigen for universal influenza vaccine development. However, epitopes induce poor immune responses. Although the use of adjuvants can overcome this obstacle, it may raise new problems. Effective antigen delivery vehicles that can function as both antigen carriers and intrinsic adjuvants are highly desired for vaccine development. Here, we report a biepitope nanovaccine that provides complete protection in mice against H3N2 virus as well as partial protection against H1N1 virus. This vaccine (3MCD-f) consists of two conserved epitopes (matrix protein 2 ectodomain (M2e) and CDhelix), and these epitopes were presented on the surface of ferritin in a sequential tandem format. Subcutaneous immunization with 3MCD-f in the absence of adjuvant induces robust humoral and cellular immune responses. These results provide a proof of concept for the 3MCD-f nanovaccine that might be an ideal candidate for future influenza pandemics.

A self-assembling nanoparticle vaccine targeting the conserved epitope of influenza virus hemagglutinin stem elicits a cross-protective immune response.

Various vaccine strategies have been developed to provide broad protection against diverse influenza viruses. The hemagglutinin (HA) stem is the major potential target of these vaccines. Enhancing immunogenicity and eliciting cross-protective immune responses are critical for HA stem-based vaccine designs. In this study, the A helix (Ah) and CD helix (CDh) from the HA stem were fused with ferritin, individually, or in tandem, yielding Ah-f, CDh-f and (A + CD)h-f nanoparticles (NPs), respectively. These NPs were produced through a prokaryotic expression system. After three immunizations with AS03-adjuvanted NPs in BALB/c mice via the subcutaneous route, CDh-f and (A + CD)h-f induced robust humoral and cellular immune responses. Furthermore, CDh-f and (A + CD)h-f conferred complete protection against a lethal challenge of H3N2 virus, while no remarkable immune responses and protective effects were detected in the Ah-f group. These results indicate that the CDh-based nanovaccine represents a promising vaccine platform against influenza, and the epitope-conjugated ferritin NPs may be a potential vaccine platform against other infectious viruses, such as SARS-COV-2.

Hemagglutinin-based DNA vaccines containing trimeric self-assembling nanoparticles confer protection against influenza.

Influenza viruses continue to threaten public health, and currently available vaccines provide insufficient immunity against seasonal and pandemic influenza. The use of recombinant trimeric hemagglutinin (HA) as an Ag provides an attractive alternative to current influenza vaccines. Aiming to develop an effective vaccine with rapid production, robust immunogenicity, and high protective efficiency, a DNA vaccine was designed by fusing influenza virus HA with self-assembled ferritin nanoparticles, denoted as HA-F. This candidate vaccine was prepared and purified in a 293-6E cell eukaryotic expression system. After BALB/c mice were immunized with 100 µg of HA-F DNA 3 times, HA-F elicited significant HA-specific humoral immunity and T cell immune responses. The HA-F DNA vaccine also conferred protection in mice against a lethal infection of homologous A/17/California/2009/38 (H1N1) virus. These results suggest that the HA-F DNA vaccine is a competitive vaccine candidate and presents a promising vaccination approach against influenza viruses.

Broad and potent bispecific neutralizing antibody gene delivery using adeno-associated viral vectors for passive immunization against HIV-1.

Broadly neutralizing antibodies (bNAbs) possess favorable safety, and passive immunization using these can prevent or control human immunodeficiency virus type 1 (HIV-1) infection. However, bNAbs generally used for monotherapy (IC80 > 5 µg/mL) have limited breadth and potency and neutralize only 70-90% of all HIV-1 strains. To address the need for broader coverage of the HIV-1 epidemic and enhance the ability of bNAbs to target HIV-1, we fused the single-chain variable antibody fragment (scFv) of bNAbs (PG9, PGT123, or NIH45-46) with full-length ibalizumab (iMab) in an scFv-monoclonal antibody tandem format to construct bispecific bNAbs (BibNAbs). Additionally, we described the feasibility of BibNAb gene delivery mediated by recombinant adeno-associated virus 8 (rAAV8) for generating long-term expression with a single injection as opposed to short-term passive immunization requiring continuous injections. Our results showed that the expressed BibNAbs targeting two distinct epitopes exhibited neutralizing activity against 20 HIV-1 pseudoviruses in vitro. After injecting a single rAAV8 vector, the expression and neutralizing activity of the BibNAbs in serum were sustained for 24 weeks. To the best of our knowledge, very few studies have been published on BibNAb gene delivery using rAAV8 vectors against HIV-1. BibNAb gene delivery using rAAV8 vectors may be promising for passive immunization against HIV-1 infection.

Expression and evaluation of porcine circovirus type 2 capsid protein mediated by recombinant adeno-associated virus 8.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide. OBJECTIVES: A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy. METHODS: In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer. RESULTS: The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2. CONCLUSIONS: The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.

Multiple Antigenic Peptide System Coupled with Amyloid Beta Protein Epitopes As An Immunization Approach to Treat Alzheimer's Disease.

Latest studies suggest that Alzheimer's disease (AD) is one of the "four big killers" that threaten the health of the elderly. AD affects about 46 million people across the world, and there is a critical need for research on new therapies for treating AD. The purpose of the present study was to develop and evaluate immunogens to elicit antibodies against the formation of amyloid beta protein (Aß), a pathological hallmark of AD, as a therapeutic strategy in AD. In this study, serial potential immunogenic epitopes were screened according to the Aß sequence. The screened linear epitopes on the Aß C-terminal fragment were coupled with either the carrier protein keyhole limpet hemocyanin (KLH) or the synthesized 4-branch peptide (MAP4). MAP4 immunogens could effectively elicit immunogenicity against Aß1-42 monomer and fiber in Balb/C mice. Furthermore, MAP4 sera could also effectively inhibit the formation of the Aß1-42 fiber. Interestingly, one of the MAP4 sera was able to depolymerize the Aß1-42 fibers that had aggregated. The monoclonal antibody, 1H7, was shown to inhibit the formation of Aß1-42 fiber. MAP4 carrier may provide benefits over current immunization strategies, as it does not induce inflammation. In conclusion, the MAP4-Aß conjugates offer a promising approach for the development of a safe and effective AD vaccine.

Humanization and directed evolution of the selenium-containing scFv phage abzyme.

According to the binding site structure and the catalytic mechanism of the native glutathione peroxidase (GPX), three glutathione derivatives, GSH-S-DNP butyl ester (hapten Be), GSH-S-DNP hexyl ester (hapten He) and GSH-S-DNP hexamethylene ester (hapten Hme) were synthesized. By a four-round panning with a human synthetic scFv phage library against three haptens, the enrichment of the scFv phage particles with specific binding activity could be determined. Three phage particles were selected binding to each glutathione derivative, respectively. After a two-step chemical mutation to convert the serine residues of the scFv phage particles into selenocysteine residues, GPX activity could be observed and determined upto 3000 U µmol-1 in the selenium-containing scFv phage abzyme which was isolated by affinity capture against the hapten Be. Also the scFv phage abzymes elicited by different antigens displayed different catalytic activities. After a directed evolution by DNA shuffling to improve the affinity to the hapten Be, a secondary library with GPX activity was created in which the catalytic activity of the selenium-containing scFv phage abzyme could be increased 17%. This study might be helpful for new haptens or antigens design to optimize the abzymes with high binding activities and might also provide a novel scheme for GPX mimic candidates for drug development.

Screening HCV genotype-specific epitope peptides based on conserved sequence analysis and B cell epitope prediction in HCV E2 region.

The high mutation rate of the hepatitis C virus (HCV) genome increases the genotype diversity and renders the detection of the virus more difficult. Therefore, prediction and assessment of highly conserved and strongly antigenic epitope polypeptide sequences have become a focus of current research. The E2 region is the target binding region of neutralizing antibodies. HCV genomics, especially the high mutation rate of E2 region sequence, makes its genotyping more and more diverse, and the detection of HCV and genotype is becoming more and more strict. In this study, four HCV B cell epitope polypeptides were constructed based on assessment of conserved sequences in the HCV E2 region and prediction of B cell epitopes, including sequences specific to genotype 1A (DC-13: 434-DTGWLAGLFYYHK-446), genotype 1B (HC-13: 434-HTGFLAALFYAKS-446), genotype 4D (NC-13: 434-NTGFLASLFYTHK-446), and a consensus sequence (FC-9: 447-FNSSGCPER-455). Epitope polypeptides combined with serum from 29 HCV-infected or 25 non-HCV-infected individuals were assayed by enzyme-linked immunosorbent assay (ELISA), and differences were analyzed by T/T' test methods in SPSS v20.0 software. Binding levels of genotype 1A, 4D, and consensus epitope polypeptides with sera of HCV-infected patients were higher than those of non-infected individuals. Moreover, binding of genotype 1B epitope polypeptides with serum of HCV 1B-infected patients was higher than that of HCV 2A-infected patients. While the screening results of HCV genotype-specific epitope polypeptides were preliminary, these findings indicated that we successfully established an HCV and genotype serological ELISA detection method. Such an approach would facilitate the discovery of epitope polypeptides which may become new antigen candidates in peptide vaccine development for the prevention of HCV infection.