A Novel Influenza Virus Hemagglutinin-Respiratory Syncytial Virus (RSV) Fusion Protein Subunit Vaccine against Influenza and RSV

Influenza A virus and respiratory syncytial virus (RSV) cause substantial morbidity and mortality afflicting the ends of the age spectrum during the autumn through winter months in the United States. The benefit of vaccination against RSV and influenza using a subunit vaccine to enhance immunity and neutralizing antibody was investigated. Influenza virus hemagglutinin (HA) and RSV fusion (F) protein were tested as vaccine components alone and in combination to explore the adjuvant properties of RSV F protein on HA immunity. Mice vaccinated with HA and F exhibited robust immunity that, when challenged, had reduced viral burden for both influenza and RSV. These studies show an enhancing and cross-protective benefit of F protein for anti-HA immunity.     

Epitope Analysis Using Anti-Oligosaccharide (G4) Monoclonal Antibody

A murine monoclonal antibody recognizing (1→6)-β-d -glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4-bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low-molecular-weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan.     

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.     

A Protective and Safe Intranasal RSV Vaccine Based on a Recombinant Prefusion-Like Form of the F Protein Bound to Bacterium-Like Particles

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.     

Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F_85–93, that carries the RSV CD8⁺ T cell epitope F_85–93 in its neuraminidase stalk. F_85–93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F_85-93 virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F_85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F_85-93 infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8⁺ T cell-mediated immunity by an influenza A virus vector expressing the RSV F_85-93 epitope.     

Protein Phosphatase PPM1G Regulates Protein Translation and Cell Growth by Dephosphorylating 4E Binding Protein 1 (4E-BP1)

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.     

Respiratory syncytial virus (RSV) G glycoprotein is not necessary for vaccine-enhanced disease induced by immunization with formalin-inactivated RSV

Following respiratory syncytial virus (RSV) challenge, mice immunized with RSV G or with formalin-inactivated RSV (FI-RSV) exhibit severe disease associated with type 2 cytokine production and pulmonary eosinophilia. This has led to the proposal that the presence of RSV G is the factor in FI-RSV that induces disease-enhancing T-cell responses. Therefore, we evaluated the role of RSV G and its immunodominant region in the induction of aberrant immune responses during FI-RSV immunization. BALB/c mice were immunized with FI preparations of wild-type (wt) RSV or recombinant RSV (rRSV) containing deletions of (i) the entire G gene, (ii) the region of the G gene encoding amino acids 187 to 197 of the immunodominant region, or (iii) the entire SH gene. After challenge, illness, RSV titers, cytokine levels, and pulmonary eosinophilia were measured. Peak RSV titers postchallenge were significantly greater in mice immunized with FI preparations of the deletion viruses than in those immunized with FI-rRSV wt, suggesting that the absence of G or SH in FI-RSV reduced its protective efficacy. Deletion of G or its epitope did not reduce illness, cytokine production, or eosinophilia relative to that in mice immunized with FI-rRSV wt. While cytokine levels and eosinophilia were similar, illness was reduced in mice immunized with SH-deleted FI-RSV. These data suggest that G-specific immune responses may be important for vaccine-induced protection and are not solely the basis for FI-RSV vaccine-enhanced illness. These data suggest that the method of RSV antigen delivery, rather than the protein composition, influences the phenotype of the induced immune responses and that RSV G should not necessarily be excluded from potential vaccine strategies.     

Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen

Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.     

Respiratory syncytial virus (RSV) infects neuronal cells and processes that innervate the lung by a process involving RSV G protein

Respiratory syncytial virus (RSV) is a primary cause of morbidity and life-threatening lower respiratory tract disease in infants and young children. Children with acute RSV bronchiolitis often develop respiratory sequelae, but the disease mechanisms are poorly understood. Mounting evidence suggests that RSV may mediate persistent infection. Using immunohistochemistry to identify RSV and RSV-infected cell types, we show that RSV infects primary neurons and neuronal processes that innervate the lungs through a process that involves RSV G protein and the G protein CX3C motif. These findings suggest a mechanism for disease chronicity and have important implications for RSV disease intervention strategies.     

Purification and Characterization of Membrane Protein (90 kDa) from Mycoplasma salivarium,Which Binds Immunoglobulin (Ig) G Fc Fragment

A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X-100 and purified by ion-exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG.     

由乙肝病毒核心蛋白(HBc-VLP)递载的流感通用疫苗Flu@uV设计构建、表征及初步活性研究

目的:构建以HBc为载体的甲型流感病毒HA和M2e流感通用疫苗(Flu@uV),利用大肠杆菌BL21(DE3)表达系统,进行初步的蛋白表达及纯化。在此基础上,构建DNA流感通用疫苗。方法:利用全基因合成的序列为模板,成功构建HA-M2e-HBc、M2e-HBc、HBc、3M2e-HBc和3HA-3M2e-HBc基因的重组质粒,并在大肠杆菌中表达,经SDS-PAGE、Western blot和电镜检测其表达。将纯化的蛋白与弗氏佐剂共同免疫小鼠,取小鼠外周血进行流式细胞分析。通过荧光分析和Western blot初步验证DNA流感通用疫苗在人源胚胎肾细胞(HEK293T)中的表达情况。结果:成功表达纯化了HA-M2e-HBc、M2e-HBc、HBc和3M2e-HBc四种蛋白,经电镜观察到30nm左右的蛋白纳米颗粒样结构。小鼠外周血流式细胞分析显示HBc和3M2e-HBc可以增加小鼠的免疫力,而HA-M2e-HBc和M2e-HBc对小鼠免疫力的提高没有影响。通过荧光检测和Western blot检测说明DNA流感通用疫苗在真核细胞中成功表达。结论:成功构建HBc与甲型流感病毒HA和M2e的病毒样颗粒,为流感通用疫苗的研制奠定了重要基础。     

Passive transfer of respiratory syncytial virus (RSV) antiserum suppresses the immune response to the RSV fusion (F) and large (G) glycoproteins expressed by recombinant vaccinia viruses.

In young infants who possess maternally derived respiratory syncytial virus (RSV) antibodies, the antibody response to RSV glycoproteins is relatively poor, despite extensive replication of RSV. In the present study, it was found that cotton rat RSV hyperimmune antiserum suppressed the antibody response to the RSV glycoproteins but not the response to vaccinia virus antigens when the antiserum was passively transferred to cotton rats prior to infection with vaccinia recombinant viruses expressing the RSV envelope glycoproteins. The cotton rats which had their immune responses suppressed by passively transferred antibodies were more susceptible to infection with RSV than were animals inoculated with control serum lacking RSV antibodies. Furthermore, many of the immunosuppressed animals infected with the vaccinia recombinant viruses developed RSV glycoprotein antibodies which had abnormally low neutralizing activities. Thus, preexisting serum RSV antibodies had dramatic quantitative and qualitative effects on the immune response to RSV glycoproteins, which may explain, in part, the poor RSV antibody response of young human infants to infection with RSV. Our observations also suggest that immunosuppression by preexisting, passively acquired RSV antibodies may constitute a major obstacle to RSV immunoprophylaxis during early infancy, when immunization is most needed.     

Recombinant Sendai virus expressing the G glycoprotein of respiratory syncytial virus (RSV) elicits immune protection against RSV

Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.     

A Single-Dose Influenza A (H5N1) Vaccine Safe and Immunogenic in Adult and Elderly Patients: an Approach to Pandemic Vaccine Development

With the ongoing pandemic of influenza A (H1N1) virus infection and the threat of high fatality rates for recent human cases of infection with highly pathogenic H5N1 strains, there has been considerable interest in developing pandemic vaccines. Here we report a randomized multicenter dose-finding clinical trial of a whole-virion, inactivated, adjuvanted H5N1 vaccine in adult and elderly volunteers. Four hundred eighty patients were randomly assigned to receive one or two doses of 3.5 μg of the vaccine or one dose of 6 or 12 μg. The subjects were monitored for safety analysis, and serum samples were obtained to assess immunogenicity by hemagglutination inhibition and microneutralization tests. The subjects developed antibody responses against the influenza A (H5N1) virus. Single doses of ≥6 μg fulfilled EU and U.S. licensing criteria for interpandemic and pandemic influenza vaccines. Except for occasional injection site pain, malaise, and fever, no adverse events were observed. We found that the present vaccine is safe and immunogenic in healthy adult and elderly subjects and requires low doses and, unlike any other H5N1 vaccines, only one injection to trigger immune responses which comply with licensing criteria. A vaccine using the same methods as those described in this report, but based on a wild-type swine-origin 2009 (H1N1) influenza A virus isolate from the United States (supplied by the CDC), has been developed and is currently being tested by our group.With the ongoing pandemic of influenza A (H1N1) virus infection and the threat of high fatality rates for recent human cases of infection with highly pathogenic H5N1 strains, there has been considerable interest in developing pandemic influenza vaccines.With new cases continuing to emerge, as of June 2009, the avian influenza A (H5N1) virus subtype has caused 433 human infections in 15 countries, as confirmed by the World Health Organization (WHO), resulting in severe illness with a high fatality rate (30). Human-to-human spread has been strongly suspected and even evidenced by statistical methods (22, 33). With new human infections continuing to develop, this subtype continues to represent a potential source of an influenza pandemic (33).Mass vaccination is the most effective approach to reduce illness and death from pandemic influenza. Therefore, vaccine producers are currently developing and assessing vaccines against H5N1 viruses (2, 14, 31). The effects of split, subvirion, and whole-virion H5N1 vaccines have been tested, with various immunogenicity results (31). Three whole-virion vaccines have been tested so far, two of which required two-dose regimens (4, 14), while a one-dose regimen with the present vaccine was found to be immunogenic in 146 adult subjects (24).The objective of the present study was to determine the safety and immunogenicity of an inactivated whole-virion vaccine against influenza A/Vietnam/1194/2004, using multiple dosing and administration schedules, for adult and elderly subjects. To date, this is the only influenza pandemic prototype vaccine trial examining single-dose regimens in elderly patients.     

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000     

Inactivated Eyedrop Influenza Vaccine Adjuvanted with Poly(I:C) Is Safe and Effective for Inducing Protective Systemic and Mucosal Immunity

The eye route has been evaluated as an efficient vaccine delivery routes. However, in order to induce sufficient antibody production with inactivated vaccine, testing of the safety and efficacy of the use of inactivated antigen plus adjuvant is needed. Here, we assessed various types of adjuvants in eyedrop as an anti-influenza serum and mucosal Ab production-enhancer in BALB/c mice. Among the adjuvants, poly (I:C) showed as much enhancement in antigen-specific serum IgG and mucosal IgA antibody production as cholera toxin (CT) after vaccinations with trivalent hemagglutinin-subunits or split H1N1 vaccine antigen in mice. Vaccination with split H1N1 eyedrop vaccine antigen plus poly(I:C) showed a similar or slightly lower efficacy in inducing antibody production than intranasal vaccination; the eyedrop vaccine-induced immunity was enough to protect mice from lethal homologous influenza A/California/04/09 (H1N1) virus challenge. Additionally, ocular inoculation with poly(I:C) plus vaccine antigen generated no signs of inflammation within 24 hours: no increases in the mRNA expression levels of inflammatory cytokines nor in the infiltration of mononuclear cells to administration sites. In contrast, CT administration induced increased expression of IL-6 cytokine mRNA and mononuclear cell infiltration in the conjunctiva within 24 hours of vaccination. Moreover, inoculated visualizing materials by eyedrop did not contaminate the surface of the olfactory bulb in mice; meanwhile, intranasally administered materials defiled the surface of the brain. On the basis of these findings, we propose that the use of eyedrop inactivated influenza vaccine plus poly(I:C) is a safe and effective mucosal vaccine strategy for inducing protective anti-influenza immunity.     

信号肽和辅助性T细胞表位增强HBV核心抗原DNA疫苗诱导的免疫应答

为增强HBVDNA疫苗的免疫效率 ,于HBV核心抗原 (HBcAg)基因 5′末端引入人IL 2信号肽和一个通用型辅助性T淋巴细胞表位基因 ,并构建成DNA疫苗 ,转染COS7细胞后经ELISA检测出分泌型HBcAg。通过肌肉注射途径分别将这种DNA疫苗和编码天然HBcAg的DNA疫苗免疫BALB/c小鼠 ,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应 ,结果表明前者诱导细胞和体液免疫应答的强度均明显超过后者 ,且更趋向于T辅助细胞 1(Th1)型免疫应答 ,故其对慢性HBV感染的治疗可能有潜在的应用价值     

Prediction of Epitope based Peptides for Vaccine Development from Complete Proteome of Novel Corona Virus (SARS-COV-2) Using Immunoinformatics

International Journal of Peptide Research and Therapeutics - COVID-19 is an infectious disease caused by a newly discovered corona virus SARS-COV-2. It is the most dangerous epidemic existing...     

Priming with Secreted Glycoprotein G of Respiratory Syncytial Virus (RSV) Augments Interleukin-5 Production and Tissue Eosinophilia after RSV Challenge

The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4⁺ T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.     

Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G(4)T(4)G(4) fragment

Using circular dichroism spectroscopy, gel electrophoresis, and ultraviolet absorption spectroscopy, we have studied quadruplex folding of RNA/DNA analogs of the Oxytricha telomere fragment, G(4)T(4)G(4), which forms the well-known basket-type, antiparallel quadruplex. We have substituted riboguanines (g) for deoxyriboguanines (G) in the positions G1, G9, G4, and G12; these positions form the terminal tetrads of the G(4)T(4)G(4) quadruplex and adopt syn, syn, anti, and anti glycosidic geometries, respectively. We show that substitution of a single sugar was able to change the quadruplex topology. With the exception of G(4)T(4)G(3)g, which adopted an antiparallel structure, all the RNA/DNA hybrid analogs formed parallel, bimolecular quadruplexes in concentrated solution at low salt. In dilute solutions ( approximately 0.1 mM nucleoside), the RNA/DNA hybrids substituted at positions 4 or 12 adopted antiparallel quadruplexes, which were especially stable in Na(+) solutions. The hybrids substituted at positions 1 and 9 preferably formed parallel quadruplexes, which were more stable than the nonmodified G(4)T(4)G(4) quadruplex in K(+) solutions. Substitutions near the 3'end of the molecule affected folding more than substitutions near the 5'end. The ability to control quadruplex folding will allow further studies of biophysical and biological properties of the various folding topologies. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 797-806, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.