Identification of two novel fowl adenovirus C-specific B cell epitopes using monoclonal antibodies against the capsid hexon protein.

The diseases associated with fowl adenovirus (FAdV) infection, such as inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HPS), and gizzard erosion (GE), were first reported in Pakistan in 1987, and subsequent outbreaks have been reported worldwide, especially in China, where severe outbreaks of HPS with high mortality from 30 to 100% were recently reported and resulted in significant economic losses to the poultry industry. The diagnosis methods of FAdVs were mostly limited to the nucleotide sequence of hexon by PCR and DNA sequencing. The aim of this study was to generate B cell epitope maps of the species- and serotype-specific hexon L1 using monoclonal antibodies (mAbs) and bioinformatics tools for the development of novel diagnostic methods. In this study, the hexon L1 (230 amino acids) was expressed and used to generate 10 mAb-expressing hybridoma cell lines against the relative protein peptide. Subsequently, we defined the linear peptide epitopes recognized by these mAbs using a series of partially overlapping peptides derived from the FAdV-C hexon protein amino acid sequence to map mAbs reactivity. Finally, a common B cell epitope (31PLAPKESMFN40) for all species FAdVs and two FAdV-C-specific epitopes (79KISGVFPNP87 and 181DYDDYNIGTT190) were identified. These mAbs and their defined epitopes may support the development of the universal or species-specific differential diagnostic methods of FAdVs.

Development and Characterization of Broadly Cross-reactive Monoclonal Antibodies Against All Known Ebolavirus Species.

As of 25 March 2015, the largest recorded outbreak of Ebola virus infection is ongoing, with almost 25 000 cases and >10 000 deaths. There are 5 genetically and antigenically distinct species within the genus Ebolavirus. Limited cross-reactivity and protection is observed between these 5 Ebolavirus species, which complicates vaccine development. However, on the basis of sequence homology between the 5 Ebolavirus species, we hypothesize that conserved epitopes are present on the viral glycoprotein (GP), which can be targeted by antibodies. In the current study, a panel of mouse monoclonal antibodies was isolated and characterized using an enzyme-linked immunosorbent assay (ELISA) to determine cross-reactivity, avidity, and competition for epitope binding; Western blot analysis was also performed. Four monoclonal antibodies were identified by ELISA as cross-reacting with the GPs of all 5 Ebolavirus species. The identification of cross-reactive antibodies that bind the GPs of all known Ebolavirus species will give us important insight into the presence of conserved epitopes on the viral GP. These data will be crucial for the development of novel therapeutics and diagnostic assays.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.

Antiviral CD8⁺ T cells cause an experimental autoimmune encephalomyelitis-like disease in naive mice.

Major histocompatibility complex class I-restricted CD8(+) cytotoxic T lymphocytes are involved in the pathogenesis of multiple sclerosis (MS) and both autoimmune, experimental autoimmune encephalomyelitis, and viral, Theiler's murine encephalomyelitis virus (TMEV) infection, animal models of MS. Following TMEV infection, certain T cell hybridomas, generated from cloned TMEV-induced CD8(+) T cells, were able to produce clinical signs of disease (flaccid hind limb paralysis) upon adoptive transfer into naive mice. Dual T cell receptors (TCR) are present on the surface of these cells as both Vß3 and Vß6 were detected by polymerase chain reaction (PCR) screening and flow cytometry and multiple Vα mRNAs were detected by PCR screening. This is the first demonstration of antiviral CD8(+) T cells having more than one TCR initiating an autoimmune disease in the natural host of the virus. We hypothesize that this is a potential mechanism for virus-induced autoimmune disease initiated by CD8(+) T cells.

Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells.

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5' end and a hepatitis delta virus ribozyme at the 3' end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10(8) copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.

Ultrastructural and immunologic characteristics of mouse x cattle xenogeneic hybridomas originating from bovine leukemia virus-infected cattle.

Nine percent of xenogeneic hybridomas originating from a bovine leukemia virus (BLV)-infected cow secreted monoclonal IgM antibodies with multispecific reactivity. Similar reactivity was evident in some antibodies with an unusually long (> 50 amino acids) third complementarity-determining region of the heavy chain. Electron microscopy of hybridomas demonstrated the presence of c-type virus particles consistent with polymerase chain reaction detection of BLV env gene. Some hybridomas contained dilated rough endoplasmic reticulum and cisternae filled with moderately electron-dense granular substance compatible with plasma cells at presecretory stage. The number of chromosomes in xenogeneic hybridomas corresponded to the sum total of mouse and bovine chromosomes. None of the hybridomas showed polyploidy. The immunochemical and genetic analysis of stable bovine immunoglobulin-secreting xenogeneic hybridomas confirms that BLV infection causes polyclonal B cell activation regardless of antigen specificity. Presence of c-type particles in hybridomas suggests that T cell-derived cytokines are not required for sustained BLV expression.

Human antigen-presenting cell/tumour cell hybrids stimulate strong allogeneic responses and present tumour-associated antigens to cytotoxic T cells in vitro.

Most tumours do not stimulate effective antitumour immune responses in vivo. In order to enhance the immunogenicity of human tumour cells, we fused a variety of tumour cell lines with an Epstein-Barr virus transformed B-lymphoblastoid cell line (EBV B-LCL) in vitro, to produce stable hybrid cells. Hybrid cell lines showed a marked increase in their ability to stimulate primary allogeneic T-cell responses in vitro, as compared with the parent tumour cells. The hybrid cells induced proliferation of naive (CD45RA+) as well as memory (CD45RO+) T lymphocytes, and both CD4+ and CD8+ subpopulations of T cells were directly stimulated. The stimulatory hybrids expressed human leucocyte antigen (HLA) class I and II, and a wide range of surface accessory molecules, including the T-cell co-stimulatory ligand molecules CD40, CD80 (B7.1) and CD86 (B7.2), the expression of which was required for optimal stimulation of T-cell responses. Fusion of the EBVB-LCL with a melanoma cell line (518.A2) yielded hybrid cells that expressed the melanoma-associated antigens MAGE-1 and MAGE-3, and presented these antigens to antigen-specific, HLA class I-restricted cytotoxic T-lymphocyte clones with greater efficiency than the parent melanoma cell line. These findings suggest that the generation of human antigen-presenting cell/tumour cell hybrids offers promise as an approach to cancer immunotherapy.

Analysis of the anti-HIV-1 activity of an anti-p17-derivative peptide (P30-52) monoclonal antibody.

The object of this study was to examine the possibility of immunotherapy using anti-human immunodeficiency virus type 1 (HIV-1) p17-derivative peptide monoclonal antibody (MAb), namely MAb 8H10. Previously we established MAb 8H10, and further characterization revealed that it inhibited the viral multiplication of the HIV-1-infected MT-4 cells, and that the inhibitory mechanism related to the decrease of p17 DNA of the infected cells. In the present study, based on the assumption that Hybridoma 8H10 (Hyb 8H10) is a source of this MAb, we examined how Hyb 8H10 influences the infected cells when the two are co-cultured using the transwell or by mixed culture. Hyb 8H10 did not influence the cell growth or viability of MT-4 cells, and MAb 8H10 was transferred to the cluster dish containing the infected cells. Furthermore, Hyb 8H10 could produce MAb 8H10 even when co-cultured with the infected MT-4 cells. However, the infectivity of the supernatant of the infected cells was temporarily enhanced when Hyb 8H10 was co-cultured without MAb 8H10, though it gradually reduced according to the increase in MAb 8H10 produced by Hyb 8H10. Though there were some problems, MAb 8H10 proved to be a strong candidate for immunotherapy against HIV.

Enumeration of antigen-presenting cells in mice infected with Sendai virus.

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.

Experiences of virus, retrovirus and retrovirus-like particles in Chinese hamster ovary (CHO) and hybridoma cells used for production of protein therapeutics.

Garnick and coworkers indicated that they experienced two independent MVM outbreaks in a period where approximately 2000 fermentations were performed, hypothesizing that such events were rare but inevitable consequences of very large scale operations. In GIs experience over the last 12 years we have seen no incidence of MVM (or any other virus) in close to 3000 fermentations, albeit at lower volumes than produced at Genentech; GI has used 250-2500L bioreactors for manufacturing whereas Genentech have reported using 100-10,000L bioreactors. Nonetheless, volumes of complex media in the same range as used at Genentech have been used at GI with no observations of viral contamination events. The reason for this is not clear. However, GI's experience in combination with experience from sub-contract testing agencies who service the majority of the biotechnology industry may call the inevitability of an MVM outbreak into question. It would appear that very few adventitious viral contaminations of cell cultures have occurred in industry in the last decade. Interestingly, the frequency of contamination events appear to be lower in CHO cells than in hybridoma cells. It should be noted, however, that these conclusions are not statistically based and the scope of the above survey was somewhat limited. RVLPs are present in both CHO and hybridoma cells. The characteristics of both are compared in Table 4. C-type particles from hybridoma cells are more abundant as a rule than those from CHO cells. Although the majority of C-type particles produced by hybridoma cells appear to be non-infective (in S+L- assays), approximately one in a million particles are competent to replicate in S+L- cells. The evidence that C-type particles can replicate in human cells has proved difficult to reproduce consistently. It is likely that replication of xenotropic hybridoma C-type particles in human cells is inefficient or restricted to only a small number of specific cell lines. C-type RVLPs from CHO cells are produced less abundantly than those from hybridoma cells and are not competent to replicate due to a defective endonuclease gene. However, over the last two decades the use of hybridoma cells and products derived from these cells has not provided any evidence of transmission of these viruses to humans; in addition they can be readily removed or inactivated. Thus, neither agent would appear to constitute a significant risk to pharmaceutical products made from their respective host cells. Nonetheless, given the difference in relative safety profiles between RVLPs from CHO and hybridoma cells it is not unreasonable to propose that safety factors (clearance factors in removal/inactivation studies in excess of the reduction of virus loads to zero) required should be less for a CHO process than for a hybridoma process.

[Production of monoclonal antibodies to the human respiratory syncytial virus]. / Poluchenie monoklonalnykh antitel k respiratorno-sintsitialnomu virusu cheloveka.

The paper describes the procedure for producing monoclonal antibodies to human respiratory asyncytial virus using hybridoma technology. Two groups of antibodies were identified in the study of their specific activity. The representatives of the first group preferably reacted with purified virus antigens. Those of the second one binded mostly to unpurified virus antigens from the cultural fluid. The antibodies of these groups appear to be different in their epitope specificity.