S. Finerty

Protective immunization against Epstein-Barr virus-induced disease in cottontop tamarins using the virus envelope glycoprotein gp340 produced from a bovine papillomavirus expression vector

Inoculation with Epstein-Barr virus (EBV) induces malignant lymphomas in the cottontop tamarin (Saguinus oedipus oedipus). This provides an experimental animal model for assessing the efficacy of candidate EBV vaccines which are intended to reduce the incidence of human tumours associated with EBV infection. Previous work has shown that experimental vaccines based on the major virus envelope glycoprotein gp340 prepared from the membranes of EBV-infected cells are effective in protecting cottontop tamarins against EBV-induced disease. However, not all purified gp340 preparations induce protective immunity against EBV lymphoma in the tamarin. In this work, cottontop tamarins were immunized with recombinant gp340, produced using a bovine papillomavirus (BPV) expression vector, and a threonyl muramyl dipeptide adjuvant formulation. Although the recombinant-derived gp340 lacked the membrane anchor sequence of authentic gp340 and was expressed in mouse cells, it was immunogenic and induced virus-neutralizing antibodies. Healthy vaccinated tamarins were protected against EBV-induced disease. The demonstration that a recombinant gp340 product is able to elicit protective immunity in the cottontop tamarin is a significant step in the development of an EBV vaccine because previously it had not been clear whether a recombinant product would have the exact tertiary structure, including the necessary carbohydrate components, to induce protective immunity. A recombinant gp340 vaccine offers various advantages over production of the authentic molecule by laborious biochemical separation, including lower cost and the absence of potentially oncogenic EBV DNA. Therefore, recombinant gp340 produced using the BPV expression vector is suitable for development as a candidate EBV vaccine for a human Phase I trial and beyond.

Targeted lymph node immunization can protect cats from a mucosal challenge with feline immunodeficiency virus.

With the rapid spread of human immunodeficiency virus (HIV) infection worldwide it is clear that effective strategies for mucosal vaccination against lentiviruses are urgently required. The aim of the present study is to determine whether protective immune responses against a mucosal challenge by feline immunodeficiency virus (FIV) can be elicited by targeting the immunization to the medial iliac lymph nodes--the principal site of migration of cells from the genital and rectal mucosa. Cats were challenged with homologous FIV via the rectal route. Targeted lymph node immunization was found to be an effective route of immunization eliciting both humoral and proliferative responses to peptide-based and fixed cell vaccines. Vaccination with fixed virus infected cells elicited protection against a cell-free mucosal FIV challenge. In addition, some cats vaccinated with fixed uninfected cells also remained uninfected following a cell-associated FIV challenge.

Epstein-Barr (EB) virus genome-containing, EB nuclear antigen-negative B-lymphocyte populations in blood in acute infectious mononucleosis.

Experiments have been performed to identify the type and size of cell infected by EB virus in the blood of acute infectious mononucleosis (IM) patients, and to investigate the nature of the infection. Virus-infected cells, recognized by their ability to give rise to lymphoblastoid cell lines when co-cultivated with foetal lymphocytes, were shown to be restricted to the B-lymphocyte population. Samples of this population from each of eight IM patients were found to be negative for EB nuclear antigen (EBNA) staining. Thereafter, fractions of IM B-lymphocytes prepared on the basis of cell size were assayed either by co-cultivation, for the incidence of virus-infected cells, or by immunofluorescence staining for the presence of cells expressing EBNA. The great majority of virus-infected cells were found in the fractions of normal sized B-lymphocytes and yet these fractions were unequivocally EBNA-negative B-cell populations in IM blood is discussed in terms of the type of infection established by EB virus in the circulation of IM patients.

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein-Barr virus (EBV)

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro re‐stimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV‐specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL‐2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual‐colour flow cytometry revealed two subpopulations, one CD4+ CD8+ population and the other CD4− CD8+. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4+ CD8+ fraction was also shown to be MHC class II‐restricted; the MHC restriction of the CD8+ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine‐induced immune protection.

Immunization of cottontop tamarins and rabbits with a candidate vaccine against the Epstein-Barr virus based on the major viral envelope glycoprotein gp340 and alum.

The Epstein-Barr virus (EBV) is associated with a range of life-threatening diseases in humans. Development of an effective vaccine has therefore been an important objective. One problem in the development of a subunit vaccine for human administration is the selection of a satisfactory adjuvant since the only one currently licensed for human use is alum, although this is not considered to be very effective. The present study demonstrated that a subunit vaccine composed of the EBV envelope glycoprotein gp340 with alum as the adjuvant did elicit protective immunity against EBV-induced lymphoma in three out of five cottontop tamarins. Furthermore, rabbits immunized with gp340/alum developed the same range of antibody responses as rabbits immunized with gp340/SAF-1, an experimental adjuvant claimed to be more effective than alum. Therefore, these results indicate that alum should be evaluated as an adjuvant as part of a human trial of a gp340-based subunit vaccine.

Mucosal immunization with experimental feline immunodeficiency virus (FIV) vaccines induces both antibody and T cell responses but does not protect against rectal FIV challenge.

Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes--the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freunds adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.

Mucosal infection and vaccination against feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology. Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes. Using a biotin-streptavidin linked immunoperoxidase technique we have detected FIV core and envelope proteins in the colonic follicle associated epithelial cells, cells within the lymphoid follice and occasional cells in the lamina propria. Further, in the intestine we have detected FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells. We have studied a group of asymptotic cats which have been rectally infected with FIV for 1 year or longer and shown an increase in the number of lamina propria CD8+ cells and greater levels of IL-2, IL-6, IL-10 and gamma-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and class I restricted cytotoxic lymphocytes (CTLs) may play a role in control of viral replication. We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with virulent virus. Cats have been immunized with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin (CT), or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes, and challenged rectally with ten mucosal cat infectious doses (MCID) of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (intraperitoneal (i.p.) versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens. However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route. Recently, we have begun to investigate if the promising results obtained with targeted lymph node (TLN) vaccination in monkeys could be reproduced in the cat. We have shown that TLN was more effective than rectal immunisation in stimulating both humoral and proliferative responses. In a preliminary study we have also been able to detect FIV specific CTLs and have observed protection from rectal challenge in four out of four cats.

Selection of monoclonal antibodies for the identification of lymphocyte surface antigens in the New World primate Saguinus oedipus oedipus (cotton top tamarin)

32 monoclonal antibodies reactive with human CD antigens were tested against tamarin peripheral blood lymphocytes, ConA blasts and lymphoblastoid B cell lines derived from tamarin cells. Reagents that cross-react with MHC class I and II, B cells (CD20, -21 and -23), monocytes (CD14) and NK cells (CD16, -56) have been identified. In addition monoclonals that cross-react with T cells (CD2, CD3), the CD4/CD8 subsets of T cells and the IL-2 receptor (CD25) are reported. A monoclonal against the beta chain of LFA-1 (CD18) cross-reacted strongly, but there was only a very poor cross-reaction with a monoclonal against the alpha chain of CD11a. Two monoclonals tested against ICAM-1(CD54) were negative.

Cellular and Molecular Events Involved in Tumour Progression in Colorectal Carcinogenesis: A Study of the Adenoma Carcinoma Sequence In Vitro

Epithelial cell lines from hereditary and sporadic adenomas have been isolated with abnormalities of chromosomes 1, 7, 14, 17, 18 and 22. Chromosome 1 may be involved in in vitro immortalization. Several stages have been described in the transformation of an adenoma cell line to anchorage independence and a possible role for sodium butyrate in tumour promotion discussed. In vitro immortality, aneuploidy, clonogenicity, resistance to sodium butyrate, ras gene activation and production of active proteinases are potential markers of tumour progression. A role for a constitutively produced tumour promoter(s) in colorectal carcinogenesis is discussed together with a possible different mechanism for adenoma formation in sporadic versus hereditary cancer patients.