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.

Molecular analysis of dengue virus attenuation after serial passage in primary dog kidney cells.

The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-O and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-O was derived. Twenty-five (0.23%) nucleotide and 10 (0.29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-O, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine. These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-O and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0.23%) nucleotide and 11 (0.32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-O strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-O and 27.

Development of a purified, inactivated, dengue-2 virus vaccine prototype in Vero cells: immunogenicity and protection in mice and rhesus monkeys.

The feasibility of a purified, inactivated dengue (DEN) vaccine made in Vero cells was explored. A DEN-2 virus candidate was chosen for production of a monotypic, purified, inactivated vaccine (PIV). Virus was harvested from roller bottle culture supernatants, concentrated, and purified on sucrose gradients. The purified virus was inactivated with 0.05% formalin at 22 degrees C. After inactivation, the virus retained its antigenicity and was immunogenic in mice and rhesus monkeys, in which it elicited high titers of DEN-2 virus-neutralizing antibody. Mice were completely protected against challenge with live, virulent virus after receiving two 0.15-microg doses of PIV. Monkeys vaccinated with three doses ranging as low as 0.25 microg demonstrated complete absence or a significant reduction in the number of days of viremia after challenge with homologous virus. These results warrant further testing and development of PIVs for other DEN virus serotypes.

Evaluation of the severe combined immunodeficient (SCID) mouse as an animal model for dengue viral infection.

Severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood lymphocytes (hu-PBL) were evaluated as an animal model for demonstrating dengue (DEN) viral infection. Reconstituted mice (hu-PBL-SCID) that demonstrated successful engraftment by the presence of serum titers of human immunoglobulin (Ig) were inoculated intraperitoneally with DEN virus serotype 1 (DEN-1). Serial blood samples were taken postinoculation and assayed for virus in C6/36 cells. The identity of all viral isolates was confirmed by an immunofluorescence antibody assay using DEN-1 monoclonal antibody. A total of six experiments were performed using different procedures of reconstitution and infection, and in three of these experiments, DEN-1 virus was recovered from the hu-PBL-SCID mice. In the first successful experiment, DEN-1 virus was recovered on postinoculation day (PID) 24 from blood, spleen, thymus, and lung tissues of one of eight hu-PBL-SCID mice. A second group of eight hu-PBL-SCID mice were inoculated with human monocytes infected in vitro with DEN-1 virus. Virus was recovered from the blood of mice between PID 15 and 23, and from lung tissue of one of these mice. In a third experiment, seven SCID mice were treated initially with anti-asialo GM1 antibody to eliminate natural killer cells, and then were injected simultaneously with a mixture of hu-PBL and DEN-1 virus. Virus was demonstrated in the blood of one mouse on PID 38, and in another mouse on PID 8, 12, 20, 24, and 36.(ABSTRACT TRUNCATED AT 250 WORDS)

A live attenuated dengue-1 vaccine candidate (45AZ5) passaged in primary dog kidney cell culture is attenuated and immunogenic for humans.

A dengue-1 candidate vaccine (45AZ5), previously found to be underattenuated in 2 volunteers, was further attenuated by passage in primary dog kidney (PDK) cell cultures. New candidate vaccines prepared from three levels of PDK-passaged virus, PDK-10, PDK-20, and PDK-27, were each injected into 9 or 10 volunteers. There was a significant, progressive decline in viremia, clinical illness, and hematologic changes from low to high PDK cell passage level. PDK-20 infected all 10 vaccinees and induced viremia in 5, transient fever in 3, symptoms that resulted in curtailed activities for < or = 1 day in 4, and neutralizing antibody in all 10, which persisted for > or = 1 year in 5 of 8 vaccinees tested. Progressive passage in PDK cell culture progressively attenuates vaccine candidate strain 45AZ5 for humans. Because passage level PDK-20 may be suitable for healthy adults at high risk of dengue fever, additional clinical trials of this strain are warranted.

Quantitative relationship between oral temperature and severity of illness following inoculation with candidate attenuated dengue virus vaccines.

The relationship between oral temperature and other parameters of illness was examined in 51 adult volunteers who were inoculated experimentally with partially attenuated candidate dengue virus vaccines. In subjects who developed clinical illness, the peak illness temperature, mean illness temperature, and peak 6:00 A.M. illness temperature all correlated positively with the total number of signs and symptoms other than fever and with a fall in the white blood cell count (the latter was the only laboratory abnormality significantly associated with clinical illness [P = .02]). Of these factors, the peak 6:00 A.M. oral temperature exhibited the strongest correlations with the two parameters used to estimate severity of illness (rxy = .58 and P < .01 for signs and symptoms; rxy = .37 and P = .01 for fall in white blood cell count).

Immunization of monkeys with baculovirus-dengue type-4 recombinants containing envelope and nonstructural proteins: evidence of priming and partial protection.

Groups of rhesus monkeys were immunized with baculovirus-dengue type-4 (DEN-4) recombinant-infected cell extracts. One recombinant contained all of the DEN-4 structural proteins and two nonstructural (NS) proteins (C-M-E-NS1-NS2a), while the other was a fusion protein containing a portion of the respiratory syncytial virus G glycoprotein and DEN-4 envelope glycoprotein (RSVG-E). Both preparations were immunogenic; all monkeys receiving either immunogen responded with the production of antivirion antibodies in enzyme immunoassays. All except one monkey receiving the recombinant b(C-M-E-NS1-NS2a) made antibodies to NS1. One monkey that received b(RSVG-E) showed the production of low levels of neutralizing antibodies. Following challenge with unmodified DEN-4 virus, seven of nine monkeys in the immunized group became infected and were viremic for a mean of 4.1 days. The control, sham-inoculated monkeys were also viremic; the mean number of days of viremia in this group was 4.7 days. The remaining monkeys in the immunized group (n = 7), although not protected, had evidence of priming. Hemagglutination inhibition antibody responses following challenge indicated an anamnestic response in this group of animals. Based on these results, it was concluded that future immunization schedules should be altered to optimize immune responses and that immunization with more potent and purified immunogens would probably result in higher seroconversion rates and antibody levels in monkeys.

Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies.

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.

Comparison of replication rates and pathogenicities between the SA14 parent and SA14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons.

The replication rates and pathogenicities of the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis (JE) virus in neurons of the mouse brain following intracerebral inoculation were compared. All the mice inoculated with the SA 14 parent strain died within one week postinoculation (p.i.), whereas all the mice inoculated with the SA 14-14-2 vaccine strains survived without showing any signs of central nervous system (CNS) involvement. The virus titers of the mouse brains inoculated with the SA 14 strain reached progressively higher levels until day 5 when the animals died. On the other hand, the virus titers of the mouse brains inoculated with the SA 14-14-2 strain persisted at low levels for several days and could not be detected after 10 days. In the routine electron microscopical study, a majority of neurons in the mouse brains inoculated with the SA 14 strain contained virions and showed characteristic cytopathological changes in connection with viral replication. In the brains inoculated with the SA 14-14-2 strain, however, we failed to find neurons containing virions or showing characteristic cytopathological changes. In the alkaline phosphatase immunostaining of paraffin-embedded sections, a majority of neurons in the brains of mice inoculated with the SA 14 strain stained positively on day 5 p.i., but only a small number of neurons in scattered small foci stained positively in the brains inoculated with the SA 14-14-2 strain. The immunogold staining of Vibratome sections also revealed the identical patterns; moreover, electron microscopical examination of the immunopositive foci of the brain inoculated with the vaccine strain revealed neurons that contained virions in dilated cisternae of rough endoplasmic reticulum (RER), indicating that the SA 14-14-2 strain also replicated, albeit poorly, in neurons. The present results showed that upon intracerebral inoculation into mice the SA 14 parent strain of JE virus grew vigorously in a large number of neurons, killing the animals, while the SA 14-14-2 vaccine strain grew poorly only in a small number of neurons without causing mortality. Possible mechanisms involved in the alteration of pathogenicity between the SA 14 parent virus and the SA 14-14-2 vaccine virus are discussed.

Large-scale purification of inactivated hepatitis A virus by centrifugation in non-ionic gradients.

Formalin-inactivated hepatitis A virus (HAV) can be purified for vaccine preparation by centrifugation in Renografin-76 (diatrizoate meglumine and diatrizoate sodium) gradients. Both continuous-flow rate-zonal and isopycnic methods were used for the separation of a major antigen component from minor antigen and host protein. The major antigen component, which appeared to contain complete virions by electron microscopy, could be recovered from gradients and accounted for approximately one third of the total antigen in the starting material. The HAV-specific purified antigen could be enriched 200-300-fold by either centrifugation procedure. The purified HAV antigen, when adsorbed to alum and inoculated into mice, was found to be highly immunogenic.

Comparative study of mouse brains infected with Japanese encephalitis virus by intracerebral or intraperitoneal inoculation.

The brains of mice infected with Japanese encephalitis (JE) virus by intracerebral inoculation (IC), intraperitoneal inoculation with sham intracerebral inoculation (IP+sIC), and intraperitoneal inoculation (IP) were studied by light and electron microscopy. The mortality rates and mean survival days were 100% and 4.8 days for the IC group, 92% and 9.0 days for the IP+sIC group, and 58% and 13.4 days for the IP group. Accordingly, the brain samples of sick mice were examined by light and electron microscopy 4 days post-inoculation (p.i.) for the IC group, 7 days p.i. for the IP+sIC group and 12 days p.i. for the IP group. In light microscopy, the mouse brains in the IC group showed little inflammatory change with only mild generalized glial-cell proliferation and mononuclear cell infiltration. In electron microscopy, however, a majority of neurons in the brain were seen to be infected with virus that replicated exclusively in the neuronal secretory system, including rough endoplasmic reticulum (RER) and the Golgi apparatus. In contrast, light microscopic observation of the brains from the IP+sIC and the IP groups showed prominent inflammatory changes with leucocytic infiltration and perivascular cuffing. Neuronal degeneration and neuronophagia were also prominent. In electron microscopy, neurons were infected in the same manner as in the IC group, but showed more advanced degenerative changes with marked cytoplasmic rarefaction and frequent neuronal disintegration. Mononuclear cells were frequently found in direct contact with degenerating and disintegrating neurons. The results showed that (a) the basic process of JE virus replication in brain neurons was present in the three groups of mice, (b) in the peripherally inoculated mice the process was accompanied by inflammatory reaction with resultant neuronal destruction, and (c) breach in the blood-brain barrier at the time of peripheral viral inoculation played an important role in the viral invasion of the CNS.

Expression of the envelope antigen of dengue virus in vaccine strains of Salmonella.

The envelope gene of dengue 4 virus (DEN) was cloned in a plasmid under the control of Escherichia coli expression signals. A clone that expressed 93% of the gene was found to be detrimental to the bacterial host. Another clone which carried only 76% of the E gene was found to be quite stable in vitro as well as in vivo. The killed recombinant bacteria induced antibodies in mice which recognized native DEN virus. Attenuated Salmonella typhimurium (SAL) strains carrying the DEN-E plasmid were tested for their efficacy as orally administered live vaccines. Protective immunization was assessed in a mouse model by immunizing three-week old BALB/c mice followed by challenge with DEN virus. It was found that these young mice were highly susceptible to the carrier SAL strains (M206 and aroA SL3261). Moreover, the SAL-infected mice were more susceptible to DEN virus challenge than control mice, suggesting that the SAL infection caused immunosuppression in these young mice.

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. II. Safety and immunogenicity in humans.

To determine safety and immunogenicity, a single 0.5 ml dose of a monovalent live-attenuated dengue (DEN) 4 (341750 Carib) vaccine was given sc to 3 groups of flavivirus nonimmune volunteers in increasing concentrations. Two recipients received 10(3) plaque forming units (PFU)/dose (1:100 dilution of stock vaccine). One remained asymptomatic, but became viremic between days 12 and 15, experienced a mild elevation of temperature (37.4 degrees C), and developed DEN-4 specific antibody. Neither recipient of the 10(4) PFU became infected. Eight volunteers then received undiluted vaccine (10(5) PFU). Viremia and antibody (neutralizing, hemagglutination inhibition, and IgM) developed in 5 of the 8 (63%). These 5 volunteers also developed a scarcely noticeable macular, blanching rash and minimal temperature elevations (37.3, 38.1, 37, 37.9, and 37.9 degrees C). Clinically insignificant decreases in total white blood cell, lymphocyte, and polymorphonuclear cell counts and an elevation in mononuclear cell counts occurred in association with viremia. This vaccine is safe, reasonably immunogenic, and suitable for further evaluation.

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. I. Pre-clinical studies.

Dengue 4 (DEN-4) virus strain 341750 Carib was modified by serial passage in primary canine kidney (PCK) cell cultures. By the 15th PCK passage, this virus was less infectious for monkeys and resulted in a significantly reduced viremia as compared to the parent DEN-4 virus. The 30th PCK passage of DEN-4 341750 Carib was non-infectious for monkeys. A vaccine prepared at the 20th PCK passage in DBS-FRhL-2 cells stimulated the production of both neutralizing and hemagglutination inhibition antibodies in monkeys; these animals were also protected against challenge with the homologous strain as well as a heterologous strain of DEN-4. An ID50 titration in monkeys resulted in a titer of greater than 10(4) plaque-forming units (PFU) for the vaccine virus and 0.5 PFU for the parent virus. Reduced monkey infectivity of this magnitude has been correlated with human attenuation in previous dengue vaccine candidates. The DEN-4 strain 341750 Carib PCK-20/FRhL-4 vaccine has been characterized and sufficiently tested to be considered for safety and immunogenicity trials in humans.

Ultrastructural changes of mouse brain neurons infected with Japanese encephalitis virus.

Ultrastructural changes of mouse brain neurons infected intracerebrally with Japanese encephalitis (JE) virus were studied. JE virus selectively infected the neurons, causing ultrastructural changes in association with viral replication in the cellular secretory system, principally involving rough endoplasmic reticulum (RER) and the Golgi apparatus. In the early phase of infection, RER of infected neurons showed hypertrophic changes, containing assembling virions within its dilated cisternae. In the later phase, the RER became cystic and degenerative and dissolved into the cytoplasm. The Golgi apparatus also contained in its saccules multiple virions, presumably transported from the RER cisternae, which were then released into the cytoplasm within coated vesicles for secretory-type exocytosis. In the process, the Golgi apparatus also fragmented and degenerated through vesiculation, vacuolation, and dispersion. Thus, the JE virus infection of neurons resulted in obliteration of RER and the Golgi apparatus, leaving behind the rarefied cytoplasm devoid of these organelles. However, destruction of the neurons themselves was not prominent and infected neurons in the later phase of infection showed some regenerative changes of these membranous organelles. The cause of death of infected animals, therefore, appeared to be extensive neuronal dysfunction rather than neuronal destruction in the CNS.

Preparation of noninfectious hepatitis A virus hemagglutinin for detecting hemagglutination inhibition antibodies.

Hepatitis A virus (HAV) harvested from infected MRC-5 cells can hemagglutinate various species of erythrocytes at acid pH (Eckels et al., 1989). Further studies revealed that the majority of the hemagglutinin (HA) in MRC-5 and BS-C-1 cells was cell-associated. A simplified procedure for preparing HAV-HA consisted of collecting infected cells in phosphate-buffered saline followed by three cycles of freeze-thawing and sonication. The fluids were clarified and stored at 4 degrees C. The analysis of HA by rate-zonal sucrose gradient centrifugation indicated that the majority of HA co-migrated with infectious virus. Complete inactivation of infectious HAV with 0.03% beta-propiolactone (BPL) did not affect HA activity, while inactivation with 0.05% formalin caused a 16-fold reduction in titer. There was no difference in HAI antibody titers when BPL-treated HA was compared to untreated HA in the hemagglutination inhibition (HAI) test.

Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection.

Five murine monoclonal antibodies (Mabs) reactive against the prM glycoproteins of DEN-3 and -4 were used to passively protect mice in vivo against lethal challenge with homologous and heterologous dengue virus serotypes. Four of the 5 prM-reactive monoclonals cross-protected mice against heterologous challenge, whereas 1 protected against challenge with only the homologous serotype. Although in vitro binding to virions was readily demonstrated, only 2 of the prM Mabs had detectable neutralizing activity. The neutralizing activity could not be enhanced by anti-mouse immunoglobulin or complement. However, 4 of the 5 prM Mabs fixed complement. This is the first report of prM-specific Mabs that are protective in mice.

Hepatitis A virus hemagglutination and a test for hemagglutination inhibition antibodies.

Like enteroviruses, hepatitis A virus (HAV) hemagglutinated various species of erythrocytes under similar conditions. HAV-specific antibodies in both acute- and convalescent-phase sera were found to inhibit hemagglutination. The HAV hemagglutination inhibition test can be used for diagnosis, epidemiological surveillance, and vaccine assessment.