Advances in protein subunit vaccines against tuberculosis.

Tuberculosis (TB), also known as the "White Plague", is caused by Mycobacterium tuberculosis (Mtb). Before the COVID-19 epidemic, TB had the highest mortality rate of any single infectious disease. Vaccination is considered one of the most effective strategies for controlling TB. Despite the limitations of the Bacille Calmette-Guérin (BCG) vaccine in terms of protection against TB among adults, it is currently the only licensed TB vaccine. Recently, with the evolution of bioinformatics and structural biology techniques to screen and optimize protective antigens of Mtb, the tremendous potential of protein subunit vaccines is being exploited. Multistage subunit vaccines obtained by fusing immunodominant antigens from different stages of TB infection are being used both to prevent and to treat TB. Additionally, the development of novel adjuvants is compensating for weaknesses of immunogenicity, which is conducive to the flourishing of subunit vaccines. With advances in the development of animal models, preclinical vaccine protection assessments are becoming increasingly accurate. This review summarizes progress in the research of protein subunit TB vaccines during the past decades to facilitate the further optimization of protein subunit vaccines that may eradicate TB.

Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody.

Glaesserella parasuis (G. parasuis.) is the etiological pathogen of Glässer's disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in G. parasuis. In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against recombinant HbpA (rHbpA) of G. parasuis SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of G. parasuis. None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among G. parasuis strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for G. parasuis. Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.

Antibody Evaluation and Mutations of Antigenic Epitopes in the Spike Protein of the Porcine Epidemic Diarrhea Virus from Pig Farms with Repeated Intentional Exposure (Feedback).

The feedback strategy, or controlled exposure of pig herd to the porcine epidemic diarrhea virus (PEDV), significantly decreased losses during a severe outbreak in late 2013 in Taiwan. However, some pig farms still suffered from recurrent outbreaks. To evaluate the association between antibody titers and clinical manifestations, sera and colostra were analyzed from one pig farm that employed the feedback strategy. Furthermore, spike (S) gene full sequences from six positive samples of two farms with and without using feedback were compared to investigate the evolution of PEDV variants circulating in pig herds. The results in this study showed that high PEDV antibody titers do not correlate with the high rate of protection from PEDV infection. In addition, repeated feedback generated the emergence of PEDV variants with unique substitutions of N537S and Y561H in the COE domain and S769F in the SS6 epitopes. These mutations indicated the pathogenetic evolution of PEDV strains existing in the cycle of the feedback method. A very strict biosecurity practice to block the routes of pathogen transfer should be followed to achieve successful control of PEDV infections in pig herds.

Chimeric Virus-like Particles of Universal Antigen Epitopes of Coronavirus and Phage Qß Coat Protein Trigger the Production of Neutralizing Antibodies.

BACKGROUND: Virus-like Particles (VLPs) are non-genetic multimeric nanoparticles synthesized through in vitro or in vivo self-assembly of one or more viral structural proteins. Immunogenicity and safety of VLPs make them ideal candidates for vaccine development and efficient nanocarriers for foreign antigens or adjuvants to activate the immune system. AIMS: The present study aimed to design and synthesize a chimeric VLP vaccine of the phage Qbeta (Qß) coat protein presenting the universal epitope of the coronavirus. METHODS: The RNA phage Qß coat protein was designed and synthesized, denoted as Qbeta. The CoV epitope, a universal epitope of coronavirus, was inserted into the C-terminal of Qbeta using genetic recombination, designated as Qbeta-CoV. The N-terminal of Qbeta-CoV was successively inserted into the TEV restriction site using mCherry red fluorescent label and modified affinity purified histidine label 6xHE, which was denoted as HE-Qbeta-CoV. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) assessment revealed the expression of Qbeta, Qbeta-CoV, and HE-Qbeta-CoV in the BL21 (DE3) cells. The fusion protein was purified by salting out using ammonium sulfate and affinity chromatography. The morphology of particles was observed using electron microscopy. The female BALB/C mice were immunized intraperitoneally with the Qbeta-CoV and HE-Qbeta-- CoV chimeric VLPs vaccines and their sera were collected for the detection of antibody level and antibody titer using ELISA. The serum is used for the neutralization test of the three viruses of MHV, PEDV, and PDCoV. RESULTS: The results revealed that the fusion proteins Qbeta, Qbeta-CoV, and HE-Qbeta-CoV could all obtain successful expression. Particles with high purity were obtained after purification; the chimeric particles of Qbeta-CoV and HE-Qbeta-CoV were found to be similar to Qbeta particles in morphology and formed chimeric VLPs. In addition, two chimeric VLP vaccines induced specific antibody responses in mice and the antibodies showed certain neutralizing activity. CONCLUSION: The successful construction of the chimeric VLPs of the phage Qß coat protein presenting the universal epitope of coronavirus provides a vaccine form with potential clinical applications for the treatment of coronavirus disease.

The genetic variability, phylogeny and functional significance of E6, E7 and LCR in human papillomavirus type 52 isolates in Sichuan, China.

BACKGROUND: Variations in human papillomavirus (HPV) E6 and E7 have been shown to be closely related to the persistence of the virus and the occurrence and development of cervical cancer. Long control region (LCR) of HPV has been shown multiple functions on regulating viral transcription. In recent years, there have been reports on E6/E7/LCR of HPV-16 and HPV-58, but there are few studies on HPV-52, especially for LCR. In this study, we focused on gene polymorphism of the HPV-52 E6/E7/LCR sequences, assessed the effects of variations on the immune recognition of viral E6 and E7 antigens, predicted the effect of LCR variations on transcription factor binding sites and provided more basic date for further study of E6/E7/LCR in Chengdu, China. METHODS: LCR/E6/E7 of the HPV-52 were amplified and sequenced to do polymorphic and phylogenetic analysis. Sequences were aligned with the reference sequence by MEGA 7.0 to identify SNP. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED 4.0. The selection pressure of E6 and E7 coding regions were estimated by Bayes empirical Bayes analysis of PAML 4.9. The HLA class-I and II binding peptides were predicted by the Immune Epitope Database server. The B cell epitopes were predicted by ABCpred server. Transcription factor binding sites in LCR were predicted by JASPAR database. RESULTS: 50 SNP sites (6 in E6, 10 in E7, 34 in LCR) were found. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. Two deletions were found between the nucleotide sites 7387-7391 (TTATG) and 7698-7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287-7288 (TG) in 97.56% (40/41) of the samples. The combinations of all the SNP sites and deletions resulted in 12 unique sequences. As shown in the neighbor-joining phylogenetic tree, except for one belonging to sub-lineage C2, others sequences clustered into sub-lineage B2. No positive selection was observed in E6 and E7. 8 non-synonymous amino acid substitutions (including E3Q and K93R in the E6, and T37I, S52D, Y59D, H61Y, D64N and L99R in the E7) were potential affecting multiple putative epitopes for both CD4+ and CD8+ T-cells and B-cells. A7168G was the most variable site (100%) and the binding sites for transcription factor VAX1 in LCR. In addition, the prediction results showed that LCR had the high probability binding sites for transcription factors SOX9, FOS, RAX, HOXA5, VAX1 and SRY. CONCLUSION: This study provides basic data for understanding the relation among E6/E7/LCR mutations, lineages and carcinogenesis. Furthermore, it provides an insight into the intrinsic geographical relatedness and biological differences of the HPV-52 variants, and contributes to further research on the HPV-52 therapeutic vaccine development.

T Cell Therapy Targeted on HLA-A02 Restricted HIV Antigen Epitopes: An Open Label Cellular Therapy Trial Using CD8+ T Cell.

Objective: To test the safety and efficacy of a T cell therapy de novo targeting HLA-A02 restricted HIV antigen epitopes. Design: This was a prospective open label clinical trial, which enrolled 28 HIV+ participants and 24 of them finished the trial. The study was publicly registered at Chinese Clinical Trial Registry, www.chictr.org.cn(ChiCTR-ICR-15005775). Method: Autologous peripheral blood mononuclear cells were co-cultured with HLA-A02 restricted HIV antigen epitopes peptides to produce cell product for this therapy. The trial was divided into five time-points with the same interval period for infusion of the cell products or monitoring parameters. Symptoms, vital signs, and blood samples were collected to analyze the safety and efficacy of this therapy. Results: Two cases of adverse effects happened during this trial in test group, which recovered without medical intervention. There was no severe adverse effect that occurred. Both symptoms and laboratory tests have no statistical significant difference between test and control group. Flowcytometry analysis showed the expression of the PD-1 and CD95 molecule on the cell surface were downregulated post-treatment in the test group. Conclusions: This autologous HIV-antigen specific effector CD8+ T cellular therapy was safe. It might have an impact on immune suppression that can provide useful reference to future cell therapy trials.

Circulation autoantibody against Lamin A/C in patients with Sjögren's syndrome.

Lamin A/C proteins are major components of nuclear laminae and were encoded by the LMNA gene. Recent studies have found that in addition to provides nuclear-membrane strength; it also regulates the gene expression. Lamin A/C has been confirmed as an autoantigen in RA, SLE and vasculitis. Anti-Lamin A/C antibodies also have been found by indirect immunofluorescence method. In this study, we used various research methods to confirm Lamin A/C is an autoantigen in Han Chinese patients with confirmed Sjögren's syndrome (SS). To further investigate the relationship between the autoimmune disease antigens, we compared the amino acid sequence of Lamin A/C epitope and several common antigens' antigenic determinant. As a result, we found that Lamin A/C has similar epitopes with U1RNP. It means that the potential relationship exist between Lamin A/C and U1RNP. Clinical data we collected also showed that anti-Lamin A/C and anti-U1RNP antibodies always appear in same serum sample. Therefore, we speculated that cross-reaction may take place between antigen and potential antigen, which have similar epitope. Then, by epitope spreading, the potential antigen can be a new autoantigen. Our study provided a new thinking for further research about the relationship between autoantigens and their development mechanism in autoimmune diseases.

A further HLA-DRB1*15 family allele - DRB1*15:112.

HLA-DRB1*15:112 differs from DRB1*15:01:01:01 by one base (282G > T) in exon 2 resulting in K65N.

Effects of polymorphic variation on the mechanism of Endoplasmic Reticulum Aminopeptidase 1.

Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) generates antigenic peptides for loading onto Major Histocompatibility Class I molecules (MHCI) and can regulate adaptive immune responses. During the last few years, many genetic studies have revealed strong associations between coding Single Nucleotide Polymorphisms (SNPs) in ERAP1 and common human diseases ranging from viral infections to cancer and autoimmunity. Functional studies have established that these SNPs affect enzyme activity resulting to changes in antigenic peptide processing, presentation by MHCI and cellular cytotoxic responses. These disease-associated polymorphisms are, however, located away from the enzyme's active site and are interspersed to different structural domains. As a result, the mechanism by which these SNPs can affect function remains largely elusive. ERAP1 utilizes a complex catalytic mechanism that involves a large conformational change between inactive and active forms and has the unique property to trim larger peptides more efficiently than smaller ones. We analyzed two of the most consistently discovered disease-associated polymorphisms, namely K528R and Q730E, for their effect on the ability of the enzyme to select substrates based on length and to undergo conformational changes. By utilizing enzymatic and computational analysis we propose that disease-associated SNPs can affect ERAP1 function by influencing: (i) substrate length selection and (ii) the conformational distribution of the protein ensemble. Our results provide novel insight on the mechanisms by which polymorphic variation distal from the active site of ERAP1 can translate to changes in function and contribute to immune system variability in humans.

Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus.

To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines.

A promising multiple-epitope recombinant vaccine against classical swine fever virus.

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine. It is caused by classical swine fever virus (CSFV), one of the members of the genus Pestivirus of the Flaviviridae family. The development of a safe and effective vaccine against the CSF is critical to pandemic control, this article shows a tandem-repeat multiple-epitope recombinant vaccine can protect pigs from CSFV challenge. That was composed as following: two copies each of glycoprotein E2 residues 693-707, 241-276 and 770-781, and two copies amino acid residues 1446-1460 of the non-structural protein NS2-3. In the challenge test, all of the swine vaccinated with Chinese vaccine strain (C-strain) were fully protected from a challenge with CSFV. However, after three successive vaccinations with the multiple-epitope recombinant vaccine, three out of five pigs were protected from challenge with CSFV (in terms of both clinical signs and viremia). These results demonstrate that multiple-epitope recombinant vaccine which carrying the major CSFV epitopes can induce a high level of epitope-specific antibodies and exhibit a protective capability that parallels induced by C-strain to a certain extent.