Yuhua Shi

Multiple antigen peptide mimetics containing gp41 membrane-proximal external region elicit broad neutralizing antibodies against human immunodeficiency virus type 1 in guinea pigs

Eliciting a broadly neutralizing antibody response against the HIV‐1 membrane‐proximal external region (MPER) mimicking the activity of 4E10 and 2F5 monoclonal antibodies remains a major challenge. In this study, two novel tetra‐branched peptide immunogens, 4E10‐ and 2F5‐MAP4, were designed and synthesized using a MAP system. Guinea pigs were immunized with either of these two synthetic immunogens emulsified in an oil‐phase adjuvant at 3‐week intervals. After four immunizations, epitope‐specific antibody responses were induced successfully, and moderate neutralizing activities against tier 1 (clades B, BC, AE) and tier 2 (clade C) HIV‐1 pseudoviruses were detectable in unfractionated sera and purified IgGs. The synthetic gp41 membrane‐proximal external region peptide mimetics, 4E10‐ and 2F5‐MAP4, assisted by an appropriate adjuvant, are promising prophylactic vaccine candidates potentially capable of eliciting broadly neutralizing antibody responses against HIV‐1. Copyright

Evaluation of the immunogenicity and protective effects of a trivalent chimeric norovirus P particle immunogen displaying influenza HA2 from subtypes H1, H3 and B

The ectodomain of the influenza A virus (IAV) hemagglutinin (HA) stem is highly conserved across strains and has shown promise as a universal influenza vaccine in a mouse model. In this study, potential B-cell epitopes were found through sequence alignment and epitope prediction in a stem fragment, HA2:90-105, which is highly conserved among virus subtypes H1, H3 and B. A norovirus (NoV) P particle platform was used to express the HA2:90-105 sequences from subtypes H1, H3 and B in loops 1, 2 and 3 of the protrusion (P) domain, respectively. Through mouse immunization and microneutralization assays, the immunogenicity and protective efficacy of the chimeric NoV P particle (trivalent HA2-PP) were tested against infection with three subtypes (H1N1, H3N2 and B) of IAV in Madin–Darby canine kidney cells. The protective efficacy of the trivalent HA2-PP was also evaluated preliminarily in vivo by virus challenge in the mouse model. The trivalent HA2-PP immunogen induced significant IgG antibody responses, which could be enhanced by a virus booster vaccination. Moreover, the trivalent HA2-PP immunogen also demonstrated in vitro neutralization of the H3 and B viruses, and in vivo protection against the H3 virus. Our results support the notion that a broadly protective vaccine approach using an HA2-based NoV P particle platform can provide cross-protection against challenge viruses of different IAV subtypes. The efficacy of the immunogen should be further enhanced for practicality, and a better understanding of the protective immune mechanism will be critical for the development of HA2-based multivalent vaccines.

A novel modified peptide derived from membrane-proximal external region of human immunodeficiency virus type 1 envelope significantly enhances retrovirus infection

Efficient gene transfer is a critical goal in retroviral transduction. Several peptides capable of forming amyloid fibrils, such as the 39‐residue semen‐derived infection‐enhancing peptide (SEVI), have demonstrated the ability to boost retroviral gene delivery. Here, a 13‐residue peptide P13 (Ac‐671NWFDITNWLWYIK683) derived from the membrane‐proximal external region of the human immunodeficiency virus type 1 (HIV‐1) gp41 transmembrane protein, together with its 16‐residue peptide derivative (P16) were found to enhance HIV‐1 infection significantly. Both peptides, P13 and P16, could form amyloid fibril structures to potently enhance HIV‐1 infectivity. Further investigations showed that both aromatic Trp residues and cationic Lys residues contributed to the enhancement of HIV‐1 infection by these two active peptides. P16 could more effectively augment HIV‐1 YU‐2 infection than SEVI, implying its potential applications as a tool in the lab to improve gene transfer rates. Copyright

Conserved stem fragment from H3 influenza hemagglutinin elicits cross-clade neutralizing antibodies through stalk-targeted blocking of conformational change during membrane fusion

Currently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments (spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin (KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide (MAP) system, strong humoral immune responses and cross-clade neutralizing activities against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design.

Elicitation of HIV-1 neutralizing antibodies by presentation of 4E10 and 10E8 epitopes on Norovirus P particles.

Eliciting efficient broadly neutralizing antibodies (BnAbs) is a major goal in vaccine development against human immunodeficiency virus type 1 (HIV-1). Conserved epitopes in the membrane-proximal external region (MPER) of HIV-1 are a significant target. In this study, Norovirus P particles (NoV PPs) were used as carriers to display conformational 4E10 and 10E8 epitopes in different patterns with an appropriate linker. Immune responses to the recombinant NoV PPs were characterized in guinea pigs and Balb/c mice and could induce high levels of MPER-binding antibodies. Modest neutralizing activities could be detected in sera of guinea pigs but not of Balb/c mice. The 4E10 or 10E8 epitopes dispersed on three loops on the outermost surface of NoV PPs (4E10-loop123 PP or 10E8-loop123 PP) elicited higher neutralizing activities than the equivalent number of epitopes presented on loop 2 only (4E10-3loop2 PP or 10E8-3loop2 PP). The epitopes on different loops of the PP were well-exposed and likely formed an appropriate conformation to induce neutralizing antibodies. Although sera of immunized guinea pigs could neutralize several HIV envelope-pseudoviruses, a vaccine candidate for efficiently inducing HIV-1 BnAbs remains to be developed.

Potential of a novel peptide P16-D from the membrane-proximal external region of human immunodeficiency virus type 1 to enhance retrovirus infection

The peptide P13 (Ac-671NWFDITNWLWYIK683-NH2), derived from the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein and its derivative P16, have been shown to significantly boost HIV-1 infectivity by forming amyloid fibrils. Here, a new modified nanofibril peptide P16-D derived from P16 was demonstrated to have an enhanced ability to promote retroviral gene transfer. Moreover, the “networks” formed by P16-D nanofibrils could effectively capture and concentrate enveloped virus by low-speed centrifugation. In addition, the captured influenza virus H1N1 could elicit a stronger immune response in mice at a lower dose than that in the absence of the nanofibrils. The results implied a potential for P16-D to improve gene transfer rates and vaccine applications.

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

Alzheimers disease (AD) is the most prevalent form of dementia worldwide and is an emerging global epidemic. Active and passive immune therapies targeting beta amyloid (Aβ) 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 Aβ 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.

Eliciting 10E8-like antibodies by the membrane proximal external region peptide of HIV-1 in guinea pigs

ObjectiveTo develop an immunotherapy for HIV that can elicit 10E8-like broadly-neutralizing antibodies in guinea pigs, using a multiple antigen peptide (MAP) system as the platform and 10E8 peptide as the epitope.ResultsThe immunogen, 10E8-MAP4, was synthetized using the MAP system. The synthetic 10E8-MAP4 was stable, and the epitopes could be exposed for recognition. In addition, the 10E8 epitope was present in an α-helical structure, which was hypothesized to aid in the generation of neutralizing antibodies. In vivo analysis showed that 10E8-MAP4 could efficiently elicit HIV binding antibodies in guinea pigs, although only weak neutralizing activities were observed.ConclusionsMultiple antigen peptide is an excellent vaccine platform for generating binding antibodies, but may elicit weak neutralizing antibodies for HIV.

Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8 in vitro and in vivo.

Despite unremitting efforts since the discovery of human immunodeficiency virus type 1 (HIV-1), an effective vaccine has not been generated. Viral vector-mediated transfer for expression of HIV-1 broadly neutralizing antibodies (BnAbs) is an attractive strategy. In this study, a recombinant adeno-associated virus 8 (rAAV8) vector was used to encode full-length antibodies against HIV-1 in 293T cells and Balb/c mice after gene transfer. The 10E8 or NIH45-46 BnAb was expressed from a single open reading frame by linking the heavy and light chains with a furin cleavage and a 2A self-processing peptide (F2A). The results showed that the BnAbs could be expressed in the 293T cell culture medium. A single intramuscular injection of rAAV8 led to long-term expression of BnAbs in Balb/c mice. The expressed antibodies in the supernatant of 293T cells and in Balb/c mice showed neutralization effects against HIV-1 pseudoviruses. Combined immunization of rAAV8 expressing 10E8 and rAAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization effects on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of rAAV8 expressing either antibody alone. Therefore, the combined immunization may be a potential vaccine approach against HIV-1.