Stanley A. Plotkin

Development of a cytomegalovirus vaccine: lessons from recent clinical trials

Cytomegalovirus-caused diseases are preventable. We believe that both neutralising antibodies and cell-mediated immunity are necessary for prevention. Of the CMV proteins, gB and pp65 are the minimum requirements in a vaccine to induce neutralising antibodies and cytotoxic T-lymphocyte (CTL) responses. Immunisation with additional proteins, e.g., gH, gN for neutralising antibodies and IE1exon 4 and pp150 for CTL responses, would strengthen protective immune responses. Approaches to development of a safe and effective cytomegalovirus (CMV) vaccine for the prevention of CMV diseases include: a) a live attenuated vaccine (Towne strain); b) recombinant constructs of the attenuated Towne and the virulent Toledo CMV strains; c) subunit glycoprotein B (gB) adjuvanted with MF59 to induce neutralising antibodies; d) phosphoprotein 65 (pp65) peptide-based vaccines to induce (CTL) for use in therapeutic vaccination; e) canarypox-CMV recombinants, e.g., ALVAC-CMV(gB) and ALVAC-CMV (pp65) to induce neutralising antibodies and CTL responses, respectively; f) DNA plasmids containing the genes for gB and pp65; g) dense bodies containing the key antigens. The attenuated Towne strain, gB/MF59, ALVAC-CMV(gB) and ALVAC-CMV(pp65) approaches have already been tested in clinical trials. The Towne vaccine induced neutralising antibodies and cell-mediated immunity (including CTLs) mitigated CMV disease in seronegative renal transplant recipients and protected against a low-dose virulent CMV challenge in normal volunteers but did not prevent infection in mothers of children excreting CMV. Immunisation with gB/MF59 resulted in high levels of neutralising antibodies in seronegative subjects. ALVAC-CMV(gB) did not induce neutralising antibodies but primed the immune system to a Towne strain challenge, while ALVAC-CMV(pp65) induced long-lasting CTL responses in all originally seronegative volunteers, with CTL precursor frequency similar to naturally seropositive individuals. These results suggest that CMV diseases can be prevented or attenuated and that a vaccine combining ALVAC-CMV(pp65) with gB/MF59 may induce sufficient CTLs and neutralising antibodies to protect against CMV diseases. Meanwhile, other approaches such as DNA peptide and dense body vaccines, should enter Phase I trials. All candidate vaccines will have to demonstrate that immunogenicity provides protection. Combined vaccines containing canarypox (ALVAC) vectors to express CMV-pp65 to induce CTLs and of subunit gB, given together with an appropriate adjuvant to induce neutralising antibodies, should be tested in a target population for the prevention of CMV infection and disease.

Human fibroblasts infected with rubella virus produce a growth inhibitor.

A protein that inhibited mitosis of normal human diploid cells was demonstrated in extracts of WI-38 cells that were infected with rubella virus and that had gone into mitotic arrest subsequent to infection. A possible mechanism for the pathogenesis of the rubella syndrome is suggested.

A rapid microneutralization assay for cytomegalovirus

A rapid, simple and reproducible microneutralization test for human cytomegalovirus is described. The results can be read in 1-2 days and the neutralization titer detected in human and guinea pig sera and in monoclonal antibody-containing supernatants is consistent with that derived by the plaque-reduction neutralization test.

Intratypic serodifferentiation of polioviruses

Abstract The intratypic serodifferentiation technique described by Wecker has been applied to the antigenic analysis of a number of attenuated and virulent strains within the same poliovirus type. By comparison of plaque inhibition produced by strain-specific immune sera, different pools of the same attenuated strain were found to be serologically identical, whereas other attenuated and virulent viruses could be distinguished antigenically from one another. The antigenic character of viruses passed in tissue culture was relatively stable, even when other markers changed considerably. After passage through laboratory animals or in the human intestine, however, some strains were stable while others were not. The applications of this test to studies of vaccination with living attenuated polioviruses are illustrated.

Induction of DNA polymerase in WI-38 and guinea pig cells infected with human cytomegalovirus (HCMV)

Abstract Infection of permissive WI-38 cells with human cytomegalovirus (HCMV) leads to the induction of DNA polymerase activities at about 20 hr postinfection (p.i.) followed by the induction of DNA synthesis at about 48 hr p.i. The infected WI-38 cells show a nuclear DNA polymerase activity that is novel in being activated by a concentration of ammonium sulfate in the in vitro reaction mixture that is inhibitory to the enzyme activity present in the cytoplasm of infected cells or the nucleus and cytoplasm of uninfected cells. The induction of DNA polymerase is blocked by an inhibitor of protein synthesis but not by an inhibitor of DNA synthesis. Induction of DNA polymerase activity was also seen in infected nonpermissive guinea pig (GP) embryo cells but this DNA polymerase is not salt-dependent. It is suggested that the novel DNA polymerase induced by HCMV infection might be an early protein(s) responsible for viral replication.

A type 3 attenuated poliovirus genetically stable after human intestinal passage.

Summary Development of a new type 3 attenuated poliovirus (WM-III) is described. Starting with the W-Fox strain, passage and clone selection at 23°C were employed to develop a strain with improved stability after human intestinal passage. The end result was a virus which had no capacity to grow at 40°C and was not virulent for monkeys injected intracerebrally. The results of tests of fecal virus strains isolated from infants fed the WM-III strain did not show the instability in relation to laboratory markers that has been reported with other attenuated type 3 viruses. We wish to thank the following individuals for help in obtaining infants for vaccination, and supervising their care: Donald Comely, M.D., Paul Gyorgy, M.D., Jane Sitnek, R. N., Walter Omans, M.D., and Elizabeth Davies, R. N., Philadelphia General Hospital; and Agnes Flack, M.D., and Ruth Lorenzo, R. N., Clinton Farms. Toby Goldschneider and Suzanne Richardson assisted with laboratory work. Dr. George Jervis performed histological examinations of the monkey central nervous system.

Ultrastructural study on the sequence of human cytomegalovirus infection in human diploid cells

The sequence of human CMV infection in WI 38 cells at high input multiplicity was studied by electron microscopy. The majority of entering virus particles were degraded within the cytoplasmic vacuoles but few particles released their capsids into the cytoplasm. Unenveloped particles with a distinct core were observed to persist over a period of 24 hours post-infection. Virus particles in the nucleus were only detectable 48 hours post-infection and thereafter. Process of formation and segregation of primary lysosome-like dense bodies was observed in association with the termination of the virus assembly cycle.

Gastroenteritis Caused By Human Rotaviruses (Serotype Three) In A Suckling Mouse Model

The pathogenic potential of human rotaviruses of serotypes 1 through 4 was evaluated in suckling mice. Oral inoculation of three different human rotaviruses of serotype 3 into 5-6 day old CD-I mice caused disease characterized by diarrhea and dehydration. The mean 50% diarrhea inducing dose (DD50) was 5xl05 pfu. Histopathological examination of small intestines revealed villus epithelial cell vacuolization localized to the distal one-third of the villus. Only Serotype 3 rotaviruses exhibited a rapid phase of viral growth in the intestine between 7 and 12 hours post-inoculation. Larger inocula of rotavirus serotypes 1, 2, and A did not cause disease or typical histopathologic changes. However, immunoperoxidase staining for rotavirus antigen was positive in all serotypes tested indicating that infection can occur without apparent disease and is not serotype specific. This convenient in-vivo model can be used to evaluate attenuation of human origin Vaccine Candidates of Serotype 3.

The characterization of IgG receptor induced by human cytomegalovirus.

Summary The IgG receptor was isolated from a lysate of human cytomegalovirus (HCMV)-infected human fibroblast cell (WI38) by the immunoprecipitation method. The IgG receptor migrated as a single peak in 7.5% of acrylamide gel elec-trophoresis and its molecular weight was 4.2 × 104 daltons. The IgG receptor was identified as glycoprotein and this protein was continuously synthesized at an almost constant rate from the early period of HCMV infection until 72 hr p.i.

HCMV Envelope Antigens Induce Both Humoral and Cellular Immunity in Guinea Pigs

Abstract Antibody and cellular immunity were measured in guinea pigs immunized with whole virion, with nucleocapsids of human cytomegalovirus or with solubilized antigens containing virus envelope proteins. All the three types of immunogens induced the production of humoral antibody as well as cytomegalovirus (CMV)-specific cellular immunity. In immunization experiments envelope antigen was as effective as immunization with whole virion.