The properties of monoclonal antibodies to HIV gag gene proteins]

Immunization of BALB/c mice with natural purified HIV antigen, fusion of spleen cells with myeloma cells and subsequent selection of hybrid clones using recombinant gag antigen of HIV gave hybridomas producing monoclonal antibodies (MCA) to HIV. The immune blotting method demonstrated that 3 clones interacted with protein p24 and 4 clones with protein p17 of HIV. Competitive EIA led to a conclusion that the resulting MCA detected at least 3 antigenic determinants in proteins, products of gag gene of HIV. The potentials of using these MCA for the detection of viral antigen in HIV-infected continuous cell lines were demonstrated.     

HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells - and not those that passed through the adult cells - remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission.     

EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with β1 and αv family integrins. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2^low recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells.     

Innate immunity including epithelial and nonspecific host factors: workshop 1B

The majority of HIV infections are initiated at mucosal sites. The oral mucosal tissue has been shown to be a potential route of entry in humans and primates. Whereas HIV RNA, proviral DNA, and infected cells are detected in the oral mucosa and saliva of infected individuals, it appears that the oral mucosa is not permissive for efficient HIV replication and therefore may differ in susceptibility to infection when compared to other mucosal sites. Since there is no definitive information regarding the fate of the HIV virion in mucosal epithelium, there is a pressing need to understand what occurs when the virus is in contact with this tissue, what mechanisms are in play to determine the outcome, and to what degree the mechanisms and outcomes differ between mucosal sites. Workshop 1B tackled 5 important questions to define current knowledge about epithelial cell-derived innate immune agents, commensal and endogenous pathogens, and epithelial cells and cells of the adaptive immune system and how they contribute to dissemination or resistance to HIV infection. Discovering factors that explain the differential susceptibility and resistance to HIV infection in mucosal sites will allow for the identification and development of novel protective strategies.     

Humoral immune response to functional regions of human cytomegalovirus glycoprotein B

Sera from patients with primary human cytomegalovirus (HCMV) infections, both acute and convalescent phase, and from HCMV-seropositive healthy subjects were analyzed to determine whether the sera would recognize antigenic domains on HCMV glycoprotein B (gB) that function in virion infectivity and spread of virus from cell to cell. The intact gB molecule, amino-terminal derivatives of different lengths, and internal deletion derivatives were expressed in eukaryotic cells and reacted by immunofluorescence with the sera. All convalescent-phase sera and most sera from healthy seropositive individuals reacted with full-length gB and with an amino-terminal derivative containing 687 amino acids (aa), gB-(1–687); approximately half of the sera recognized an amino-terminal derivative of 447 aa, gB-(1–447), and one-third reacted with the shortest deletion derivative of 258 aa, gB-(1–258). Of the acute-phase sera, 77% recognized intact gB and gB-(1–687), 32% recognized gB-(1–447), and 14% recognized gB-(1–258). Deletion of aa 548 to 618 dramatically reduced the percentage of reactive sera, whereas deletion of aa 411 to 447 had a minor impact on reactivity of sera. To investigate the epitope specificity of human antibodies to gB, we carried out competition experiments between human sera with neutralizing activity and selected monoclonal antibodies (mAbs) to conformational epitopes on gB. We found that antibodies in human sera preclude syncytium formation in UB15-11 glioblastoma cells constitutively expressing gB and compete with certain murine mAbs that block virus entry into cells and transmission of infection from cell to cell. Our results show that HCMV-immune human sera contain antibodies to functional regions on gB, and the abundance of these antibodies in convalescent-phase sera suggests that they may play a central role in limiting dissemination of virus in the host. J. Med. Virol. 52:451–459, 1997. © 1997 Wiley-Liss, Inc.     

Changes in the gene expression of the hepatitis B virus in PLC-PRF-5 cells of human hepatocarcinoma during their acquisition of drug resistance]

Mutant clones of hepatocarcinoma cell line PLC-PRF-5 containing integrated hepatitis B virus genome were derived by multistep selection for resistance to some toxic agents. These clones were found to display various stages of cell differentiation according to growth rate, ability to synthesize alpha-fetoprotein, to grow in semisolid medium and to cloning in agar. In the majority of the clones with higher stages of differentiation the expression of HBsAg gene was demonstrated to be increased as compared to the original line, and in some of these clones the expression of silent HBcAg was activated.     

pH-dependent fusion of tick-borne encephalitis virus with artificial membranes

Summary pH-dependent fusion of TBE virus with artificial membranes was effective at slightly acidic pH with maximum at 6.4. The influence of various changes in E protein conformation on fusion process was studied.     

HIV infection and specific mucosal immunity: workshop 4B

Most HIV infections are transmitted across mucosal epithelium. An area of fundamental importance is understanding the role of innate and specific mucosal immunity in susceptibility or protection against HIV infection, as well as the effect of HIV infection on mucosal immunity, which leads to increased susceptibility to bacterial, fungal, and viral infections of oral and other mucosae. This workshop attempted to address 5 basic issues-namely, HIV acquisition across mucosal surfaces, innate and adaptive immunity in HIV resistance, antiviral activity of breast milk as a model mucosal fluid, neutralizing immunoglobulin A antibodies against HIV, and progress toward a mucosal vaccine against HIV. The workshop attendants agreed that progress had been made in each area covered, with much recent information. However, these advances revealed how little work had been performed on stratified squamous epithelium compared with columnar epithelium, and the attendants identified several important biological questions that had not been addressed. It is increasingly clear that innate immunity has an important biological role, although basic understanding of the mechanisms of normal homeostasis is still being investigated. Application of the emerging knowledge was lacking with regard to homeostatic mucosal immunity to HIV and its role in changing this homeostasis. With regard to breast milk, a series of studies have demonstrated the differences between transmitters and nontransmitters, although whether these findings could be generalized to other secretions such as saliva was less clear. Important progress toward an oral mucosal HIV vaccine has been made, demonstrating proof of principle for administering vaccine candidates into oral lymphoid tissues to trigger anti-HIV local and systemic immune responses. Similarly, experimental data emphasized the central role of neutralizing antibodies to prevent HIV infection via mucosal routes.     

Human hybridoma neutralizing antibodies to the herpes simplex virus]

Human hybridoma cells producing monoclonal antibodies to human herpes simplex virus were generated. The antibody produced by the hybrid cells interacted with herpes virus types I and II in enzyme immunoassay and complement fixation test, had neutralizing activity both in vivo and in vitro. These human monoclonal antibodies may be used for immunotherapy of herpes virus infection.