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

OBJECTIVE: To 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. RESULTS: The 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. CONCLUSIONS: Multiple antigen peptide is an excellent vaccine platform for generating binding antibodies, but may elicit weak neutralizing antibodies for HIV.

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 antimicrobial peptide derived from membrane-proximal external region of human immunodeficiency virus type 1.

With increasing microbial drug resistance worldwide, antimicrobial peptides (AMPs) are considered promising alternatives to addressing this problem. In this study, a series of synthetic peptides were designed based on the membrane-disrupting properties of the membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) envelope protein. The peptide AP16-A was found to exhibit the most effective antimicrobial activities against both Gram-negative and Gram-positive bacteria. The minimal bactericidal concentration (MBC) of AP16-A ranged from 2 µg/ml to 16 µg/ml. AP16-A had no detectable cytotoxicity in various tissue cultures and a mouse model. Furthermore, results of confocal fluorescence microscopy and the SYTOX Green uptake assay indicated that AP16-A killed Gram-negative bacteria by the combined effects of relatively slow membrane permeabilization and interaction with an intracellular target, while it killed Gram-positive bacteria by a fast membrane permeabilization process, which achieved relatively more rapid bacterial killing kinetics. The results of this study support the potential use of AP16-A as an AMP.

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.