Phase 1 studies of Walter Reed Army Institute of Research candidate attenuated dengue vaccines: selection of safe and immunogenic monovalent vaccines.

We describe the results of initial safety testing of 10 live-attenuated dengue virus (DENV) vaccine candidates modified by serial passage in primary dog kidney (PDK) cells at the Walter Reed Army Institute of Research. The Phase 1 studies, conducted in 65 volunteers, were designed to select an attenuated vaccine candidate for each DENV serotype. No recipient of the DENV candidate vaccines sustained serious injury or required treatment. Three vaccine candidates were associated with transient idiosyncratic reactions in one volunteer each, resulting in their withdrawal from further clinical development. Increasing PDK cell passage of DENV-1, DENV-2, and DENV-3 candidate vaccines increased attenuation for volunteers, yet also decreased infectivity and immunogenicity. This effect was less clear for DENV-4 candidate vaccines following 15 and 20 PDK cell passages. Only one passage level each of the tested DENV-2, -3, and -4 vaccine candidates was judged acceptably reactogenic and suitable for expanded clinical study. Subsequent studies with more recipients will further establish safety and immunogenicity of the four selected vaccine candidates: DENV-1 45AZ5 PDK 20, DENV-2 S16803 PDK 50, DENV-3 CH53489 PDK 20, and DENV-4 341750 PDK 20.

Atypical antibody responses in dengue vaccine recipients.

Eight of 69 (12%) healthy adult volunteers vaccinated with monovalent live-attenuated dengue virus (DENV) vaccine candidates had atypical antibody responses, with depressed IgM:IgG antibody ratios and induction of high-titer hemagglutination-inhibiting and neutralizing (NT) antibodies to all four DENV serotypes. These features suggested flavivirus exposure prior to DENV vaccination, yet no volunteer had a history of previous flavivirus infection, flavivirus vaccination, or antibody to flaviviruses evident before DENV vaccination. Moreover, production of antibody to DENV by atypical responders (AR) was not accelerated compared with antibody responses in the 61 flavivirus-naive responders (NR). Further evaluation revealed no differences in sex, age, race, DENV vaccine candidate received, or clinical signs and symptoms following vaccination between AR and NR. However, viremia was delayed at the onset in AR compared with NR. A comparative panel of all AR and five randomly selected NR found flavivirus cross-reactive antibody after vaccination only in AR. Unexpectedly, six of eight AR had NT antibodies to yellow fever virus (YFV) > 1:10 before vaccination while NR had none (P = 0.04). The AR also universally demonstrated YFV NT antibody titers > or = 1:160 after DENV vaccination, whereas four of five NR failed to seroconvert (P = 0.02). Yellow fever virus priming broadens the antibody response to monovalent DENV vaccination. The effect of flavivirus priming on the clinical and immunologic response to tetravalent DENV vaccine remains to be determined.

Short report: absence of protective neutralizng antibodies to West Nile virus in subjects following vaccination with Japanese encephalitis or dengue vaccines.

Protection of individuals against West Nile (WN) encephalitis is an emerging concern in the United States and Europe. We investigated whether immunization with licensed inactivated Japanese encephalitis (JE) vaccine or experimental live attenuated dengue vaccines resulted in induction of cross-neutralizing antibodies against WN virus. Protective neutralizing antibody titers to WN virus were not detected in any volunteer despite successful immunization to related flaviviruses. Vaccination against JE or dengue is unlikely to prevent WN virus infection but may still protect against disease.

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers.

A randomized, controlled, double-blinded study was conducted to determine safety and immunogenicity of five live attenuated dengue vaccines produced by Aventis Pasteur (AvP). The study was completed with 40 flavivirus non-immune volunteers: five recipients of each monovalent (dengue-1, dengue-2, dengue-3, or dengue-4) vaccine, ten recipients of tetravalent (dengue-1, dengue-2, dengue-3, and dengue-4) vaccine, and ten recipients of vaccine vehicle alone. All vaccines were administered in a single subcutaneous dose (range, 3.6-4.4 log(10) plaque forming units). No serious adverse reactions occurred in volunteers followed for 6 months after vaccination. Five vaccine recipients developed fever (T > or = 38.0 degrees C), including four tetravalent vaccinees between days 8 and 10 after vaccination. Dengue-1, dengue-2, dengue-3, or dengue-4 vaccine recipients reported similar frequency of mild symptoms of headache, malaise, and eye pain. Tetravalent vaccinees noted more moderate symptoms with onset from study days 8-11 and developed maculopapular rashes distributed over trunk and extremities. Transient neutropenia (white blood cells < 4000/mm3) was noted after vaccination but not thrombocytopenia (platelets < 100,000/mm3). All dengue-3, dengue-4, and tetravalent vaccine recipients were viremic between days 7 and 12 but viremia was rarely detected in dengue-1 or dengue-2 vaccinees. All dengue-2, dengue-3, and dengue-4, and 60% of dengue-1 vaccine recipients developed neutralizing and/or immunoglobulin M antibodies. All tetravalent vaccine recipients were viremic with dengue-3 virus and developed neutralizing antibodies to dengue-3 virus. Seven volunteers also had multivalent antibody responses, yet the highest antibody titers were against dengue-3 virus. The AvP live attenuated dengue virus vaccines are safe and tolerable in humans. The live attenuated tetravalent dengue vaccine was most reactogenic, and preferential replication of dengue-3 virus may have affected its infectivity and immunogenicity.

Limited potential for transmission of live dengue virus vaccine candidates by Aedes aegypti and Aedes albopictus.

To evaluate the transmission risk of four live dengue (DEN) vaccine candidates developed by the U.S. Army (DEN-1, 45AZ5 PDK 20; DEN-2, S16803 PDK 50; DEN-3, CH53489 PDK 20; and DEN-4, 341750 PDK 20), we tested 3,010 Aedes aegypti and 1,576 Aedes albopictus mosquitoes blood-fed on 21 volunteers who had been administered one of the four vaccine candidates or the licensed yellow fever (YF) vaccine (17D). We used an indirect immunofluorescence assay (IFA) to detect DEN or YF viral antigen in the heads of mosquitoes. Corresponding to the lack of a detectable viremia among volunteers inoculated 8-13 days previously with live DEN-1 or DEN-2 vaccine candidates, only six mosquitoes developed disseminated infections after feeding on these volunteers. These six mosquitoes included 4 of 247 Ae. albopictus fed on volunteers inoculated with the DEN-1 vaccine candidate and 2 of 528 Ae. aegypti fed on volunteers inoculated with the DEN-2 vaccine candidate. Infection was confirmed in each of these IFA-positive mosquitoes by isolating infectious virus from the mosquito's body in Vero-cell culture. None of the 1,252 or the 969 mosquitoes fed on DEN-3 or DEN-4 recipients, respectively, were infected. Overall, dissemination rates in Ae. albopictus and Ae. aegypti were low. Dissemination rates were 0.5%, 0.3%, < 0.1%, and < 0.1% for the DEN-1 through DEN-4 vaccine candidates, respectively. Because of the observed low dissemination rates, it is unlikely that these vaccine viruses would be transmitted under natural conditions.

Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in Escherichia coli are protected against lethal dengue virus infection.

A gene fragment encoding the C-terminal 204 amino acids (AA) from the structural envelope glycoprotein (E) and the N-terminal 65 AA from non-structural protein one (NS1) of dengue type 2 virus (DEN-2) was expressed in Escherichia coli (E. coli) as a fusion protein with staphylococcal protein A. The recombinant fusion protein was purified and analysed for its antigenicity, its immunogenicity and its ability to protect mice against lethal challenge with live DEN-2 virus. The recombinant protein was found to be reactive with anti-DEN-2 polyclonal and monoclonal antibodies. Mice immunized with the purified fusion protein made anti-DEN-2 antibodies measured by the hemagglutination-inhibition (HI) and neutralization (N) tests, and were protected against lethal challenge with DEN-2 virus administered by intracranial inoculation.

The dengue viruses.

Dengue, a major public health problem throughout subtropical and tropical regions, is an acute infectious disease characterized by biphasic fever, headache, pain in various parts of the body, prostration, rash, lymphadenopathy, and leukopenia. In more severe or complicated dengue, patients present with a severe febrile illness characterized by abnormalities of hemostasis and increased vascular permeability, which in some instances results in a hypovolemic shock. Four distinct serotypes of the dengue virus (dengue-1, dengue-2, dengue-3, and dengue-4) exist, with numerous virus strains found worldwide. Molecular cloning methods have led to a greater understanding of the structure of the RNA genome and definition of virus-specific structural and nonstructural proteins. Progress towards producing safe, effective dengue virus vaccines, a goal for over 45 years, has been made.

Protection of mice against yellow fever virus encephalitis by immunization with a vaccinia virus recombinant encoding the yellow fever virus non-structural proteins, NS1, NS2a and NS2b.

Recent evidence of a protective immune response to the flavivirus non-structural protein, NS1, has suggested its incorporation into possible recombinant vaccines. The region of the 17D yellow fever virus (YFV) genome encoding the C terminus of envelope glycoprotein and extending to the N terminus of non-structural protein NS3 (NS1-NS2a-NS2b; nucleotides 2030 to 4940) was expressed in vaccinia virus and physical and immunogenic properties of the NS1 moiety were studied. Recombinant NS1 protein, and native YFV NS1, was detected at the surface of infected cells by immunofluorescence and by immune cytolysis after treatment with anti-NS1 antibody and complement. NS1 was also detected in the extracellular medium as a secreted form. Recombinant NS1 was immunoprecipitated as a single protein of approximately the same size as native 17D YFV NS1. Unboiled, both recombinant and native NS1 formed polymers of high Mr. Immunization of mice with this recombinant vaccinia virus stimulated production of non-neutralizing, complement-fixing cytolytic antibody and conferred partial protection against lethal intracerebral inoculation of mice with live 17D YFV.

Cell surface expression of yellow fever virus non-structural glycoprotein NS1: consequences of interaction with antibody.

Among antibodies to flaviviral proteins only those directed at the virion envelope protein (E) or the non-structural glycoprotein NS1 are known to confer protection. To investigate the possible role of complement-mediated cytolysis (CMC) in protection we measured the capacity of anti-NS1, or E monospecific serum or monoclonal antibodies to bind to yellow fever virus (YFV)-infected cells and of anti-NS1 or E serum to sensitize them to CMC. Although both anti-NS1 and anti-E antibody bound to YFV-infected cells, CMC was observed only with anti-NS1 antibody. Greater binding by anti-NS1 antibody suggested the presence of larger amounts of NS1 than E associated with the cell membrane. Using the cell membrane-impermeable, cross-linking reagent BS3, cell surface NS1, but not E, was detected as a homopolymer, a form in which bound antibody might be expected to activate complement more efficiently. Peak titres of progeny virus were reduced 10- to 100-fold when infected cells were treated with complement-fixing, anti-NS1 monoclonal antibody or monospecific, anti-NS1 rabbit serum and complement. Taken together these results are consistent with the hypothesis that CMC subserved by anti-NS1 antibody provides an alternative to direct neutralization of virus in the protective immune response to flaviviral infection.

Functional and antigenic domains of the dengue-2 virus nonstructural glycoprotein NS-1.

The gene coding for the nonstructural glycoprotein of dengue-2 virus was cloned, sequenced, and expressed in Escherichia coli. There was about 70% conservation at the amino acid level with dengue serotypes 1 and 4 suggesting an important common function for this protein. Conserved hydrophobic domains were found both before the amino-terminus and at the carboxy-terminus, consistent with transmembrane roles. Evidence for at least partial translocation of NS-1 through the inner membrane of E. coli was found. Also conserved were two signals for N-linked glycosylation located near the middle of NS-1. Various regions of NS-1 were tested for antigenicity with mouse and rabbit polyclonal and mouse monoclonal antibodies. The mouse polyclonal antibodies, made against a crude dengue-infected mouse brain immunogen, reacted most strongly with N-terminal regions of NS-1, whereas, the rabbit antiserum, made against purified NS-1 protein, reacted strongest with C-terminal regions. These findings suggest that immunogen presentation or species differences could be important. Although most of the monoclonals appeared to be unreactive in Western blots with expressed NS-1 proteins, two appeared to react strongly; the region from amino acid (a.a.) 273 to a.a. 346 was required for antibody binding. This region, located adjacent to the two conserved C-terminal hydrophobic domains, is highly charged and contains 5 of the 10 conserved cysteine residues of NS-1.

Use of temperature-sensitive mutants to study the morphogenesis of poliovirus.

Three temperature-sensitive (ts) mutants of poliovirus (type 1 Mahoney) were isolated after nitrous acid treatment and characterized as phenotypically RNA+. When cells were infected at 37 degrees with two of the three RNA+ ts mutants (ts109 and ts739), reduced levels of 14 S particles were synthesized. One RNA+ mutant (ts520) synthesized significant amounts of viral 14 S particle subunits. All of the mutants synthesized reduced amounts of procapsids and virions at 37 degrees. At 39.5 degrees, with all three ts mutants, the production of all virus-related particles in infected cells was markedly suppressed. Isoelectric focusing of the viral-related particles produced at 37 degrees by the ts mutants and electrophoretic analysis of their structural polypeptides revealed the following: (i) ts739 synthesized an altered VP0 polypeptide and produced 14 S particles with an altered isoelectric point; (ii) ts109 produced 14 S particles with a normal pI but containing what appeared to be an altered VP1; (iii) ts520 produced normal 14 S particles as demonstrated by their pI, the electrophoretic behavior of their constituent structural polypeptides in SDS-PAGE, their ability to self-assemble, and their ability to form procapsid-like structures when incubated in extracts from wild-type (wt) virus-infected cells. However, ts520-infected cells contained few, if any, procapsids and extracts made therefrom were unable to assemble ts520 or wt 14 S particles into detectable amounts of pI 6.8 empty capsids. These and other findings are consistent with ts739 (and probably ts109) possessing an altered structural protein and ts520 being mutant in its morphopoietic factor.

Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids.

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.

Differences between poliovirus empty capsids formed in vivo and those formed in vitro: a role for the morphopoietic factor.

Empty capsid species formed from the self- and extract-mediated assembly of poliovirus type 1 14S particles in vitro and procapsids isolated from virus-infected cells were subjected to isoelectric focusing in charge-free agarose gels. The empty capsid formed in the self-assembly reaction had an isoelectric point (pI) of 5.0, whereas procapsids and extract-assembled empty capsids focused at pH 6.8. Unreacted 14S particles focused at pH 4.8 to 5.0. The sedimentation coefficient (s20,w) and density of the empty capsid species were also determined. Procapsids had a density in CsCl of 1.31 g/cm3, whereas empty capsids formed by self- or extract-mediated assembly had a density of 1.29 g/cm3. Both extract-assembled empty capsids and procapsids had an s20,w of 75S, whereas self-assembled empty capsids had an s20,w of 71S. Self-assembled empty capsids were not converted to pI 6.8 empty capsids by incubation with poliovirus-infected HeLa cell extracts. The dissociated polypeptides of self-assembled empty capsids (pI 5.0) and procapsids (pI 6.8) behaved identically when analyzed by isoelectric focusing in the presence of 9 M urea and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These results suggest that infected cell extracts possess a factor that influences the final conformation of the empty shell (pI 6.8, 75S) formed from 14S particles and that this influences is exerted at the initiation step or during the polymerization reaction. A small amount of this activity (less than or equal to 20% of infected extracts) was detected in uninfected cells; the significance of this remains unknown.

Serodiagnosis of hepatitis B virus infection by antibody to core antigen.

In a military population antibody to hepatitis B core antigen (anti-HBc) was found in 39% of acute hepatitis cases negative for hepatitis B surface antigen (HBS Ag) and in 96% of HBs Ag-positive cases. Persistence of antibody to HBs Ag (anti-HBs) in convalescent-phase sera was significantly greater (P less than 0.001) in individuals with acute HBs Ag-positive hepatitis B than in patients with clinical HBs Ag-negative hepatitis B. The prevalence of anti-HBc in the absence of HBs Ag, anti-HBs, and clinical disease was 3.2% in this military population. In longitudinal studies of hepatitis B infection, the presence of anti-HBc preceded anti-HBs and improved the ability to determine the onset of sublicnical infection. Anti-HBc is a useful serologic marker for the study of the epidemiology of hepatitis B and improves the efficiency of detection of hepatitis B virus infection.