Selected regulatory and scientific topics for candidate rotavirus vaccine development.

Various aspects of the development of rotavirus vaccine candidates are discussed. As is true with other vaccines, comprehensive testing must be done to detect the possible presence of adventitious agents in the vaccine and seed preparations. Consideration must also be given to other biologic materials that come in contact with the vaccine preparation during production to prevent the introduction of contaminants. The clinical testing of rotavirus vaccines from early safety and immunogenicity studies through final efficacy studies is also discussed. Issues surrounding coadministration of investigational rotavirus vaccines with US-licensed vaccines are ideally addressed before initiation of efficacy trials. Other subjects discussed are identification of correlates of protection, multivalent vaccines, foreign efficacy trials, safety data, and statistical considerations. Sponsors of investigational vaccines are urged to contact the Food and Drug Administration for guidance during the development process, especially before the investigational new drug application and pivotal efficacy trial stages.

Increased immunogenicity and protective efficacy in outbred and inbred mice by strategic carboxyl-terminal truncation of Japanese encephalitis virus envelope glycoprotein.

We constructed recombinant vaccinia viruses expressing the full-length envelope (E) glycoprotein of Japanese encephalitis virus (JEV) or a strategically truncated E glycoprotein, approximately 80% of the N-terminal sequence, and compared their antigenic structure and protective immunity in mice. The truncation site in the JEV E glycoprotein sequence corresponds to the position that had been shown to increase the immunogenicity of dengue type 4 or type 2 virus E glycoprotein. Analysis of the JEV E glycoprotein in recombinant virus-infected cells showed that C-terminally truncated E retains an antigenic structure similar to that of the full-length E glycoprotein. The full-length JEV E glycoprotein was detected predominantly intracellularly, while a small fraction (< 2%) was present on the cell surface. On the other hand, the truncated 80% E glycoprotein exhibited an alteration in the intracellular transport pathway resulting in increased accumulation (10-25%) on the cell surface and secretion (6-10%) into the medium. The C-terminally truncated E glycoprotein induced a greater antibody response and a higher level of protective immunity than did the full-length E glycoprotein in outbred CD-1 mice as well as in two strains of inbred mice that differ in their resistance to intraperitoneal (ip) JEV infection. In the case of outbred CD-1 and inbred C57/Bl mice, which possess a dominant autosomal genetic locus that controls resistance to a high dose of ip infection of JEV or the capacity to acquire resistance to intracerebral JEV infection, truncated E glycoprotein induced a higher titer of JEV neutralizing antibodies.

Development of cross-reactive antibodies to plasminogen during the immune response to dengue virus infection.

The four serotypes of dengue virus (a mosquito-borne flavivirus) cause an acute febrile illness (dengue fever) or a more prolonged illness with plasma leakage resulting in hypovolemia (dengue hemorrhagic fever). Hemorrhage may accompany either. Epidemiologic data suggest a role for dengue antibodies in pathogenesis. Computer analysis revealed a 20-residue region of similarity in amino acid sequence between the dengue type 4 envelope glycoprotein (E) and a family of clotting factors, including plasminogen, the prime mediator of fibrinolysis. By use of synthetic peptides in ELISA, E antibodies that potentially bind plasminogen were detected in 75% of 40 Thai patients acutely infected with dengue virus type 1, 2, 3, or 4. Plasminogen cross-reactivity of dengue antibodies was shown to be specific for the related sites in E and plasminogen. The dengue E sequence with similarity to plasminogen is largely conserved within the currently known flavivirus E sequences. However, 15 Thai patients hospitalized for illness caused by Japanese encephalitis virus (a flavivirus not associated with hemorrhage) did not develop plasminogen-cross-reactive antibodies, and this finding correlated with failure of Japanese encephalitis virus antibodies to bind to the plasminogen-cross-reactive site in E.

Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus.

Non-neutralizing, serotype-specific anti-NS1 monoclonal antibodies partially protected passively immunized mice from lethal dengue 2 virus intracerebral challenge. There was no apparent correlation between complement-fixing activity and protective capacity among individual anti-NS1 monoclonal antibodies. Immunization with specific combinations of non-protective or partially protective antibodies resulted in prolonged survival or reduced mortality. Solid protection, equal to that achieved after immunization with neutralizing polyclonal antibody, was achieved only with an antibody pair which individually fixed complement to high titre with homologous virus. Some groups of mice had increased morbidity after immunization with combinations of protective monoclonal antibodies that bind to overlapping epitopes. These results may affect the design of recombinant dengue vaccines which may require the inclusion of serotype-specific antigenic domains.

Variation in dengue type 2 viruses isolated in Bangkok during 1980.

Dengue type-2 viruses isolated in metropolitan Bangkok during 1980 (Bangkok/80) were characterized by oligonucleotide fingerprinting, restriction enzyme (RE) mapping and antigenic analysis using monoclonal antibody probes. Of 10 isolates analysed by oligonucleotide fingerprinting, nine were very closely related, showing 72.5% to 91.4% oligonucleotide homology. One isolate (D80-141) produced a distinctly different fingerprint (55.7% to 58.0% homology) and was less related to other Bangkok/80 dengue-2 virus isolates than to a 1964 Bangkok isolate (16681). RE mapping conducted on complementary dsDNA prepared from three Bangkok/80 isolates, strain 16681 and the prototype New Guinea C strain confirmed that D80-141 was genetically distinct. On antigenic analysis, only one of 22 monoclonal antibody probes produced against representative 1980 Bangkok dengue-2 isolates, D80-100 and D80-141, was able to distinguish between these virus strains. Monoclonal antibody 47-10/10, prepared using D80-100 virus and directed at the NS1 non-structural glycoprotein, had a significantly lower (100-fold) solid phase radioimmune assay endpoint titre for D80-141 antigen than for D80-100 antigen. By the indirect immunofluorescence assay, 47-10/10 had lower antibody endpoint titres against D80-141, the NGC strain and 13 (12%) of 110 Bangkok/80 isolates than to a control antibody preparation. These results suggest that strain D80-141 represents a second minor topotype of dengue-2 which was circulating concurrently with the major endemic topotype in Bangkok in early 1980.

Topological mapping of unique epitopes on the dengue-2 virus NS1 protein using monoclonal antibodies.

Monoclonal antibodies were produced against two distinct Thai dengue-2 (DEN-2) virus strains isolated in 1980 from dengue haemorrhagic fever patients. Nine of 36 hybridomas produced monoclonal IgG antibodies which reacted in radioimmune precipitation assays with the NS1 non-structural protein (42,000 mol. wt.) from DEN-2-infected C6/36 (Aedes albopictus) cells. The virus specificity of NS1-reactive monoclonal antibodies was determined by indirect immunofluorescence assays using LLC-MK2 cells infected with either the Thai 1980 DEN-2 isolates, prototype DEN viruses (four serotypes), Japanese encephalitis (JE), Murray Valley encephalitis, West Nile, Wesselbron or Tembusu viruses. Eight of the monoclonal antibody preparations were DEN-2-serotype specific. One preparation defined a special serological relationship between DEN-2 and JE viruses. Four preparations had detectable complement fixation titres using Thai DEN-2 virus antigen. Six spatially unique epitopes were identified using competitive binding assays.

Identification and typing of herpes simplex virus types 1 and 2 by monoclonal antibodies, sensitivity to the drug (E)-5-(2-bromovinyl)-2'-deoxyuridine, and restriction endonuclease analysis of viral DNA.

With development of antiviral drugs, the need to identify a virus as to drug sensitivity becomes increasingly of importance. The compound (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) has been shown to be much more inhibitory to the replication of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus as opposed to herpes simplex virus type 2 (HSV-2). We have typed over 170 isolates, using an immunofluorescent technique and sensitivity to the drug BVDU. These results were then compared to the typing of isolates by analysis of viral DNA after restriction endonuclease digestion (EcoRI). Without exception the results were in agreement between the monoclonal antibody results and sensitivity to the drug BVDU. Furthermore, the typing with monoclonal antibodies was also in excellent agreement with the DNA analysis. Only those isolates inhibited with BVDU showed DNA characteristics of HSV-1 and reacted only with the S-200 antibody. On the other hand, those isolates which reacted with the monoclonal antibody S-141 were insensitive to BVDU, and again this was in agreement with the DNA analysis. These results could provide the basis for developing a diagnostic test using the two monoclonal antibodies to type either isolates or direct smears and to use the results as a basis for possible drug therapy.