Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells.

CD40 ligation has been shown to promote antigen-presenting functions of dendritic cells, which express CD40 receptor. Here we reported significantly altered biodistribution and immune responses with the use of CD40-targeted adenovirus. Compared with unmodified adenovirus 5, the CD40-targeted adenovirus following intravenous administration (i.v.) resulted in increased transgene expressions in the lung and thymus, which normally do not take up significant amounts of adenovirus. Intradermal injection saw modified adenovirus being mainly processed in local draining lymph nodes and skin. Following intranasal administration (i.n.), neither unmodified nor targeted viruses were found to be in the liver or spleen, which predominantly took up the virus following i.v. administration. However, inadvertent infection of the brain was found with unmodified adenoviruses, with the second highest gene expression among 14 tissues examined. Importantly, such undesirable effects were largely ablated with the use of targeted vector. Moreover, the targeted adenovirus elicited more sustained antigen-specific cellular immune responses (up to 17-fold) at later time points (30 days post boosting), but also significantly hampered humoral responses irrespective of administration routes. Additional data suggest the skewed immune responses induced by the targeted adenoviruses were not due to the identity of the transgene but more likely a combination of overall transgene load and CD40 stimulation.

Selective induction of tumor-associated antigens in murine pulmonary vasculature using double-targeted adenoviral vectors.

Targeted therapies directed to tumor-associated antigens are being investigated for the treatment of cancer. However, there are few suitable animal models for testing the ability to target these tumor markers. Therefore, we have exploited mice transgenic for the human coxsackie and adenovirus receptor (hCAR) to establish a new model for transient expression of human tumor-associated antigens in the pulmonary vasculature. Systemic administration of Ad in hCAR mice resulted in an increase in transgene expression in the lungs compared to wild-type mice, as determined using a luciferase reporter gene. To reduce transgene expression in the liver, the predominant organ of ectopic Ad localization and transgene expression following systemic administration, we utilized the endothelial-specific flt-1 promoter, which resulted in a further increased lung-to-liver ratio of luciferase expression. Administration of an adenoviral vector encoding the tumor-associated antigen carcinoembryonic antigen (CEA) under transcriptional control of the flt-1 promoter resulted in selective expression of this antigen in the pulmonary vasculature of hCAR mice. Feasibility of targeting to expressed CEA was subsequently demonstrated using adenoviral vectors preincubated with a bifunctional adapter molecule recognizing this tumor-associated antigen, thus demonstrating utility of this transient transgenic animal model.

Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells.

A promising approach to immunotherapy involves the loading of dendritic cells (DCs) with genetic material to facilitate sustained expression of a relevant antigen in this population of potent antigen presenting cells (APC). Viral vectors such as adenovirus (Ad) have been used for this purpose. Existing methods for DC infection are limited by lack of specificity and a requirement for DC exposure to high viral doses. Targeting of Ad to DCs with bispecific antibodies has significantly augmented levels of transgene expression. Genetic fusion of the extracellular portion of coxsackievirus-adenovirus receptor (CAR) to cell-specific ligands has also proved successful in targeting Ad to cells of interest. We report here the production and primary characterization of a new fusion protein comprising the ecto-domain of CAR connected to a single chain antibody (scFv) G28-5 against human CD40 present on the surface of DCs. We demonstrate that the fusion protein (CAR/G28) specifically interacts with both recombinant Ad fiber knob and the ecto-domain of human CD40 in a binding assay (ELISA). Finally, we show that the CAR/G28 fusion protein promotes highly efficient transduction of DCs of both rhesus monkey and human origin.

Epitopes in the interacting regions of beta-dystroglycan (PPxY motif) and dystrophin (WW domain).

The dystroglycan gene produces two products from a single mRNA, the extracellular alpha-dystroglycan and the transmembrane beta-dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via beta-dystroglycan, the WW domain of dystrophin interacting with a PPxY motif in beta-dystroglycan. A panel of four monoclonal antibodies (MANDAG1-4) was produced using the last 16 amino acids of beta-dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four beta-dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb, MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first beta-strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin-dystroglycan complex, highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ.

[Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus]. / Monoklonal'nye antitela k virusu Ebola: poluchenie, kharakteristika i izuchenie perekrestnoi reaktivnosti s virusom Marburg.

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.

Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen.

A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.

Identification of antigenic sites on three hepatitis C virus proteins using phage-displayed peptide libraries.

A novel approach to screening phage-displayed peptide libraries has been used to identify hepatitis C virus (HCV) core, NS4 and NS5 sequences, which are antigenic in humans. Two random peptide libraries were used for screening using a mixture of HCV-positive sera or individual antibodies to core, NS3, NS4, and NS5 HCV proteins affinity-purified from this mixture. Sequencing of 56 selected phage clones resulted in 28 different peptide sequences and identification of seven antigenic regions, three in the core protein (19-26, 34-49, and 73-83), three in the NS4 (1681-1693, 1712-1718, and 1726-1736) and one in the NS5 protein (2251-2260). No NS3-specific peptides were identified. The immune response to core, NS4 and NS5 proteins includes a variety of linear determinants whereas epitopes on the investigated part of NS3 protein appear to be conformation-dependent.

Glycoproteins E2 of the Venezuelan and eastern equine encephalomyelitis viruses contain multiple cross-reactive epitopes.

Enzyme immunoassay (EIA) with sixty types of monoclonal antibodies (MAbs) was used to study cross-reactive epitopes on the attenuated and virulent strains of the Eastern equine encephalomyelitis (EEE) and Venezuelan equine encephalomyelitis (VEE) viruses. All three structural proteins of the EEE and VEE viruses were demonstrated to have both cross-reactive and specific antigenic determinants. The glycoprotein E1 of EEE and VEE viruses possesses three cross-reactive epitopes for binding to MAbs. The glycoprotein E2 has a cluster of epitopes for 20 cross-reacting MAbs produced to EEE and VEE viruses. Cross-reactive epitopes were localised within five different sites of glycoprotein E2 of VEE virus and within four sites of that of the EEE virus. There are no cross-neutralising MAbs to the VEE and EEE viruses. Only one type of the protective Mabs was able to cross-protect mice against lethal infection by the virulent strains of the VEE and EEE viruses. Eight MAbs blocked the hemagglutination activity (HA) of both viruses. Antigenic alterations of neutralising and protective sites were revealed for all attenuated strains of the VEE and EEE viruses. Comparative studies of the E2 proteins amino acid sequences show that the antigenic modifications observed with the attenuated strains of the VEE virus may be caused by multiple amino acid changes in positions 7, 62, 120, 192 and 209-213. The escape-variants of the VEE virus obtained with cross-reactive MAbs 7D1, 2D4 and 7A6 have mutations of the E2 protein at positions 59, 212-213 and 232, respectively. Amino acid sequences in these regions of the VEE and EEE viruses are not homologous. These observations indicate that cross-reactive MAbs are capable of recognising discontinuous epitopes on the E2 glycoprotein.

Design of immunogens as components of a new generation of molecular vaccines.

Three new approaches to design effective immunogens are considered. At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface. EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting. In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system. Chimerical particles of HBcAg containing epitope of the VEE virus were obtained. A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained. The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1. We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle. The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells. Mice immunized with TBI showed humoral and cellular immune responses to HIV-1. Anti-TBI antibodies displayed HIV-1 neutralizing activity. These new approaches offer promise in the development of new effective vaccines.

Design of four-helix bundle protein as a candidate for HIV vaccine.

To be efficient, a synthetic vaccine should contain different T and B cell epitopes of human immunodeficiency virus (HIV) antigens, and the B epitope regions in the vaccine and in the HIV should be conformationally similar. We have suggested previously the construction of vaccines in the form of a protein with a predetermined tertiary structure, namely a four-alpha-helix bundle. Antigenic determinants of cellular and humoral immunity are blocks for the vaccine design. From experimentally studied HIV-1 T and B cell epitopes, we constructed a sequence of a four-helix protein TBI (T and B cell epitopes containing immunogen). The gene of the protein was synthesized and the protein was produced in C600 Escherichia coli cells under recA promoter from Proteus mirabelis. CD spectroscopy of the protein demonstrated that 30% of amino acid residues adopt an alpha-helical conformation. Mice immunized with TBI have shown both humoral and cellular immune responses to HIV-1. The obtained data show that the design of TBI was successful. The synthesized gene structure makes possible further reconstruction and improvement of the protein vaccine structure.

Analysis of the hemagglutination activity domains of the Venezuelan equine encephalomyelitis and eastern equine encephalomyelitis viruses.

The hemagglutination (HA) domains of the Venezuelan equine encephalomyelitis (VEE) and the eastern equine encephalomyelitis (EEE) viruses providing the interaction of virions and red blood cells were studied with the use of a panel of 17 hemagglutination inhibition (HI) monoclonal antibodies (MAbs). A highly conserved domain (C domain) forming alphavirus-group-reactive MAbs was identified in the E2 protein of the VEE and EEE viruses. These MAbs inhibited HA of the western equine encephalomyelitis, Semliki Forest, Sindbis, Getah, Aura, Chikungunya and Pixuna viruses. The involvement of amino acid residues 59 and 232 in the formation of the C region was demonstrated by sequencing the gene encoding the E2 protein of three escape variants of the VEE virus.

[The peptide mapping of the E2-2 and E2-6 sites of the E2 glycoprotein in the Venezuelan equine encephalomyelitis virus]. / Kartirovanie s pomoshch'iu peptidov saitov E2-2 i E2-6 glikoproteina E2 virusa venesuél'skogo éntsefalomielita loshadei.

Nine peptides were synthesized for detailed mapping of VEE virus E2-2 and E2-6 sites responsible for the formation of protective antibodies that neutralize the virus and block hemagglutination. The sequence of the peptides over-lapped the regions of amino acid residues 30-75 and 202-250 of VEE virus E2 protein in which antigenic mutations caused by monoclonal antibodies to E2-2 and E2-6 sites had been mapped. None of the synthesized peptides reacted in the enzyme immunoassay with a panel of 17 Mabs to VEE virus E2 protein. However, eight peptides reacted with polyclonal antiviral serum and two of them elicited antiviral antibody production. The E2-2 site might be associated with amino acid residues 30-45, and the region of E2 residues 57-62 in which antigenic mutations are observed is not a linear type antigenic determinant, but participates in the formation of antigenic determinants of the conformational type. The mapping of residues 202-250 demonstrated that all the peptides in this region were well recognized by polyclonal antiviral serum. The residues 235-240 were shown to form a linear epitope which provided a crossover between VEE and EEE viruses and was not recognized by 19 types of monoclonal antibodies cross-reacting with VEE and EEE viruses.

[The antigenic structure of the eastern equine encephalomyelitis virus studied by using monoclonal antibodies]. / Issledovanie antigennoi struktury virusa vostochnogo éntsefalomielita loshadei s pomoshch'iu monoklonal'nykh antitel.

Thirty-three monoclonal antibodies (Mabs) interacting with the structural proteins of Eastern equine encephalomyelitis (EEE) virus were prepared. The mutual arrangement of the antigenic sites on the E1 and E2 glycoproteins was studied by competitive radioimmunoassay. At least four nonoverlapping sites were found on the E1. The E2 glycoprotein contained at least seven partially overlapping antigenic sites. Mabs to the sites E2-2 and E2-3 neutralized viral infectivity and blocked hemagglutination. Mabs to the site E2-1 blocked hemagglutination. Mabs to sites E2-2, 3, and 7 protected mice against lethal infection although the protective Mabs to sites E2-2b and E2-7 did not neutralize the virus. The antibodies to the other three sites of E2 and to all sites of E1 did not have any biological activity. The experimental results indicate the dominant role of E2 in antiviral immunity, over 98% of the observed protective effect being associated with the E2-2 site.

[A monoclonal antibody study of the receptor area of the Venezuelan encephalitis virus]. / Izuchenie retseptornoi oblasti virusa venesuél'skogo éntsefalita loshadei pri pomoshchi monoklonal'nykh antitel.

The receptor region for virus-cell interaction in Venezuelan equine encephalomyelitis (VEE) and Eastern equine encephalomyelitis (EEE) viruses was studied using a panel of 17 monoclonal antibodies (MCA). They were able to block agglutination of goose erythrocytes. The dominant role of glycoprotein E2 in the formation of viral receptor for EEE and VEE viruses was demonstrated. Competitive radioimmunoassay identified three antigenic sites in this region. These sites were also responsible for virus neutralization. MCAs to these sites protected outbred mice against lethal infection. The presence of a highly conservative region in VEE (site E2-3) and EEE (site E2a) which produced cross-reacting antibodies blocking hemagglutination of Western equine encephalomyelitis, Semliki Forest, Sindbis, Getah, Aura, Chikungunya, and Pixuna viruses was established. A hypothesis is suggested concerning the existence of similar regions for the entire alphavirus genus, and the role of this region in virus-cell interaction.

[Study of the antigenic structure of the E1 glycoprotein of the Venezuelan equine encephalomyelitis virus using monoclonal antibodies]. / Izuchenie antigennoi struktury glikoproteina E1 virusa venesuél'skogo éntsefalomielia loshadei s pomoshch'iu monoklonal'nykh antitel.

The collection of eight rat and mouse hybridomas secreting the high affinity monoclonal antibodies to glycoprotein E1 of the Venezuelan equine encephalomyelitis has been obtained. The antigenic structure of E1 protein has been studied with the use of these antibodies for the strains Trinidad, TC-83 and 230 of the virus. Antigenic map of glycoprotein E1 based on competition radioimmunoanalysis is proposed. Five sites are mapped including eight epitopes binding monoclonal antibodies. Antibodies to sites E1-1, E1-3 and E1-5 are crossreactive in interaction with the virus of Venezuelan equine encephalomyelitis, while antibodies to site E1-5 interact also with the virus of tick-borne encephalitis. Antibodies to site E1-1 possess the protective effect and lack the neutralizing effect in tissue cultures. Antibodies to all sites of E1 protein are devoid of ability to neutralize the Venezuelan equine encephalitis virus.

Investigation of antigenic structure of attenuated and virulent Venezuelan equine encephalomyelitis virus by means of monoclonal antibodies.

A comparative study of the antigenic structure of virulent strains and attenuated vaccine strains of Venezuelan equine encephalomyelitis virus (VEEV) by means of monoclonal antibodies has made it possible to investigate the antigenic structure of the envelope glycoproteins E1 and E2, and to specify their role in the development of antiviral immunity. On the E1 glycoprotein there are five nonoverlapping antigenic sites consisting of eight epitopes that are recognized by monoclonal antibodies; six sites consisting of twenty epitopes were found on the E2 glycoprotein. The monoclonal antibodies against four sites protect the animals from lethal infection with the virulent strain, Trinidad donkey. Out of the thirteen epitopes identified as being responsible for antiviral immunity, three are changes in the TC-83 strain, and six belong to two sites in the strain 230. The results obtained indicate the necessity for further improvement of the available vaccine preparations against this dangerous infectious disease.

[The antigenic structure of glycoprotein E2 in the Venezuelan equine encephalomyelitis virus studied using rat monoclonal antibodies]. / Izuchenie antigennoi struktury glikoproteida E2 virusa venesuél'skogo éntsefalomielita loshadei s pomoshch'iu krysinykh monoklonal'nykh antitel.

A collection of 21 rat hybridomas secreting high-affinity monoclonal antibodies to Venezuelan equine encephalomyelitis (VEE) virus was generated. Using a panel of 15 monoclonal antibodies to glycoprotein E2, the antigenic structure of this protein of VEE strains TC-83 and 230 was studied. A competitive radioimmunoassay suggested a new map of the antigenic structure of glycoprotein E2 in which 5 sites including 11 epitopes of monoclonal antibody binding were distinguished. Antibody to E2-2 site neutralized virus infectivity and blocked hemagglutination test and antibody to E2-3 site could only block hemagglutination. Antibodies to other E2 protein sites lacked any biological activity.

[Monoclonal antibodies cross-reacting with the tick-borne encephalitis virus and with the Venezuelan equine encephalomyelitis virus]. / Monoklonal'nye antitela, perekrestno reagiruiushchie s virusom kleshchevogo éntsefalita i virusom venesuél'skogo éntsfalomielita loshadei.

Employment of radioimmunoassay led to the demonstration of serological crossing between tick-borne encephalitis (TBE) virus and Venezuelan equine encephalomyelitis (VEE) virus. Using hybridoma technology, three hybridomas were produced secreting monoclonal antibodies (MAb) cross-reacting with these two viruses. With MAb, the epitope of binding of these antibodies was shown to be located on protein E of TBE virus and protein E1 of VEE virus. Despite the low percentage (14%) of homology of amino acid sequences of these proteins, 12 areas with homology from 24% to 63% were demonstrated. Considering conservative replacements, homology of these areas was 53%-75%. The assumed existence of some of these areas in alpha-helical conformation may explain the observed immunological crossing.