ABSTRACT
Investigation of targeting inhibitors of Aß aggregation, heme-Aß peroxidase-like activity and efficient detectors of Aß aggregation, are of therapeutic value and diagnostics significance for the treatment of Alzheimer's disease (AD). Due to the complex pathogenesis of AD, theranostics treatment with multiple functions are necessary. Herein we constructed the NIR absorption property of gold nanorods (GNRs) loaded with single chain variable fragment (scFv) 12B4 and thermophilic acylpeptide hydrolase (APH) ST0779 as a smart theranostic complex (GNRs-APH-scFv, GAS), which possesses both rapid detection of Aß aggregates and NIR photothermal treatment that effectively disassembles Aß aggregates and inhibits Aß-mediated toxicity. Methods: We screened targeting anti-Aß scFv 12B4 and thermophilic acylpeptide hydrolase as amyloid-degrading enzyme, synthesized GAS gold nanorods complex. The GAS was evalued by Aß inhibition and disaggregation assays, Aß detection assays, Aß mediated toxicity assays in vitro. In vivo, delaying Aß-induced paralysis in AD model of Caenorhabditis elegans was also tested by GAS. Results: In vitro, GAS has a synergistic effect to inhibit and disassociate Aß aggregates, in addition to decrease heme-Aß peroxidase-like activity. In cultured cells, treatment with GAS reduces Aß-induced cytotoxicity, while also delaying Aß-mediated paralysis in CL4176 C.elegans model of AD. Furthermore, the photothermal effect of the GAS upon NIR laser irradiation not only helps disassociate the Aß aggregates but also boosts APH activity to clear Aß. The GAS, as a targeting detector and inhibitor, allows real-time detection of Aß aggregates. Conclusion: These results firstly highlight the combination of scFv, APH and nanoparticles to be theranostic AD drugs. Taken together, our strategy provides a new thought into the design of smart compounds for use as efficiently therapeutic and preventive agents against AD. Moreover, our design provides broad prospects of biomedical strategy for further theranostics application in those diseases caused by abnormal protein.
Subject(s)
Alzheimer Disease/therapy , Hyperthermia, Induced/methods , Nanoconjugates/chemistry , Peptide Hydrolases/metabolism , Phototherapy/methods , Single-Chain Antibodies/chemistry , Theranostic Nanomedicine/methods , Alzheimer Disease/diagnostic imaging , Amyloid beta-Peptides/metabolism , Animals , Caenorhabditis elegans , Cell Line, Tumor , Enzyme Stability , Gold/chemistry , Humans , Infrared Rays , Nanotubes/chemistry , Peptide Hydrolases/chemistry , Peptide Hydrolases/radiation effects , Proteolysis , Smart Materials/chemistryABSTRACT
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.
Subject(s)
Alzheimer Disease , Alzheimer Vaccines/immunology , Amyloid beta-Peptides/immunology , Vaccines, Synthetic/immunology , Animals , Epitopes/immunology , Female , Mice , Mice, Inbred BALB CABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Blood-Brain Barrier/metabolism , Brain/metabolism , Cell Membrane Permeability/drug effects , Endothelium, Vascular/metabolism , Insulin/pharmacology , Transcriptome/drug effects , ATP-Binding Cassette Transporters/genetics , Amyloid beta-Peptides/metabolism , Biological Transport , Blood-Brain Barrier/drug effects , Brain/cytology , Brain/drug effects , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Gene Expression Regulation , Humans , Hypoglycemic Agents/pharmacology , In Vitro Techniques , Models, BiologicalABSTRACT
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.
Subject(s)
Enzyme Inhibitors/chemistry , Influenza A Virus, H1N1 Subtype/drug effects , Influenza, Human/drug therapy , Neuraminidase/chemistry , Catalytic Domain/drug effects , Enzyme Inhibitors/pharmacology , Humans , Influenza A Virus, H1N1 Subtype/enzymology , Influenza, Human/virology , Neuraminidase/antagonists & inhibitors , VirionABSTRACT
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.
ABSTRACT
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.
Subject(s)
Epitopes, B-Lymphocyte/genetics , Genotype , Hepacivirus/immunology , Hepatitis C/immunology , Peptides/genetics , Viral Envelope Proteins/genetics , Viral Hepatitis Vaccines/immunology , Adult , Aged , Antibodies, Neutralizing/metabolism , Conserved Sequence/genetics , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Female , Humans , Immune Sera , Male , Middle Aged , Protein Binding , Vaccines, SubunitABSTRACT
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.
Subject(s)
Adjuvants, Immunologic/pharmacology , Cancer Vaccines/pharmacology , Melanoma, Experimental/drug therapy , Peptide Fragments/immunology , Poly I-C/pharmacology , Polysorbates/pharmacology , Skin Neoplasms/drug therapy , Squalene/pharmacology , Survivin/immunology , Animals , Cancer Vaccines/chemical synthesis , Cancer Vaccines/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Cytotoxicity, Immunologic/drug effects , Epitopes , Female , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Immunogenicity, Vaccine , Lymphocyte Activation/drug effects , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice, Inbred C57BL , Peptide Fragments/chemical synthesis , Poly I-C/immunology , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Squalene/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Burden/drug effectsABSTRACT
Influenza viruses are a major public health threat worldwide. The influenza hemagglutinin (HA) plays an essential role in the virus life cycle. Due to the high conservation of the HA stem region, it has become an especially attractive target for inhibitors for therapeutics. In this study, molecular simulation was applied to study the mechanism of a small molecule inhibitor (MBX2329) of influenza HA. Behaviors of the small molecule under neutral and acidic conditions were investigated, and an interesting dynamic binding mechanism was found. The results suggested that the binding of the inhibitor with HA under neutral conditions facilitates only its intake, while it interacts with HA under acidic conditions using a different mechanism at a new binding site. After a series of experiments, we believe that binding of the inhibitor can prevent the release of HA1 from HA2, further maintaining the rigidity of the HA2 loop and stabilizing the distance between the long helix and short helices. The investigated residues in the new binding site show high conservation, implying that the new binding pocket has the potential to be an effective drug target. The results of this study will provide a theoretical basis for the mechanism of new influenza virus inhibitors.
Subject(s)
Antiviral Agents/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Influenza A Virus, H1N1 Subtype/chemistry , Molecular Dynamics Simulation , Protein Structure, SecondaryABSTRACT
A novel water-soluble fluorescent probe FLS2 based on the thiooxofluorescein derivative has been firstly designed and synthesized. UV-vis absorption and fluorescence spectra studies showed that the FLS2 as a colorimetric and ratiometric fluorescent probe exhibited high selectivity and sensitivity towards Hg2+, which was mainly attributed to the special binding with the receptor unit accompanied with the spirolactam ring-opening progress. In addition, the probe FLS2 could be used as a naked-eye indicator for Hg2+ with reversible response. It displayed approximate 37-fold fluorescent enhancement at 529nm in the presence of only 2.0 equiv. Hg2+ and the detection limit was calculated at about 39nM. What's more, cellular imaging experiment revealed that the sensor had excellent biocompatibility and low cytotoxicity that could be utilized for monitoring Hg2+ in living cells.
Subject(s)
Chemistry Techniques, Analytical/instrumentation , Colorimetry/methods , Fluorescein/chemistry , Fluorescent Dyes/chemistry , Mercury/analysis , Mercury/chemistry , Cell Survival , HeLa Cells , Humans , Hydrogen-Ion Concentration , Optical Imaging , Spectrometry, FluorescenceABSTRACT
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.
Subject(s)
Cell Membrane/chemistry , HIV Antibodies/immunology , HIV-1/immunology , Peptides/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/blood , Chromatography, High Pressure Liquid , Circular Dichroism , Enzyme-Linked Immunosorbent Assay , Guinea Pigs , Immunization , Neutralization Tests , Peptides/chemistry , Protein BindingABSTRACT
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.
Subject(s)
AIDS Vaccines/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , Animals , Blotting, Western , Dependovirus , Enzyme-Linked Immunosorbent Assay , Female , Genetic Vectors , HEK293 Cells , HIV-1/immunology , Humans , Mice , Mice, Inbred BALB C , Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/immunology , Immunogenicity, Vaccine , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H3N2 Subtype/genetics , Influenza B virus/genetics , Influenza Vaccines/immunology , Norovirus/genetics , A549 Cells , Animals , Antibodies, Viral/blood , Chimerin Proteins/administration & dosage , Chimerin Proteins/genetics , Chimerin Proteins/immunology , Cross Protection , Dogs , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Female , Humans , Immunization , Immunoglobulin G/blood , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza B virus/immunology , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/prevention & controlABSTRACT
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.
Subject(s)
Anti-Infective Agents/pharmacology , HIV-1/chemistry , Peptides/pharmacology , Amino Acid Sequence , Animals , Anti-Infective Agents/isolation & purification , Female , Mice , Mice, Inbred BALB C , Peptides/isolation & purificationABSTRACT
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.
Subject(s)
Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Orthomyxoviridae Infections/immunology , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Combinatorial Chemistry Techniques , Computer Simulation , Conserved Sequence/genetics , Crystallography, X-Ray , Epitope Mapping , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Female , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Membrane Fusion , Mice , Mice, Inbred BALB C , Protein Conformation , Sequence AlignmentABSTRACT
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.