A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of dengue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified once in these cells to yield a titer of 6.3 log 10 PFU/ml. The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebrally. This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control group received only the vaccine diluents. All monkeys (except the control group) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log 10 focus forming units (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log 10 FFU/ml) was detected in any vaccinated group, whereas all animals in the placebo control group developed viremia. All vaccinated monkeys developed neutralizing antibodies in a dose-dependent response. In another experiment, viremia and production of neutralizing antibodies were determined in YF-immune monkeys that received either ChimeriVax-D2 or a wild-type dengue-2 virus. Low viremia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue-2-immunized animals became viremic. All of these animals were protected against challenge with a wild-type dengue-2 virus, whereas all YF-immune monkeys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous in vitro passage in VeroPM cells. The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice, and the sequence of the inserted prME genes were unchanged after 18 passages in Vero cells. The high replication efficiency, attenuation phenotype in mice and monkeys, immunogenicity and protective efficacy, and genomic stability of ChimeriVax-D2 justify it as a novel vaccine candidate to be evaluated in humans.

We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477-5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ϳ7.5 log 10 PFU/ml in Vero cells, were not neurovirulent in 3-to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/ DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.

No abstract

Respiratory syncytial virus (RSV), the major cause of lower respiratory tract disease in infants, is thought to infect the upper airways before spreading to the lower respiratory tract. A rhesus monkey model of RSV infection after upper airway inoculation was used to test the protective effect of intranasal treatment with HNK20, a mouse monoclonal IgA antibody against RSV F glycoprotein. HNK20 was administered once daily for 2 days before RSV challenge and 4 days after challenge. Treatment with 0.5 mg/kg HNK20 reduced viral shedding in the nose, throat, and lungs by 3-4 log10/mL (P < or = .002). All monkeys developed RSV neutralizing antibody in serum, even in the absence of detectable viral replication. Neutralizing concentrations of monoclonal antibody remained in nasal secretions for > 1 day after treatment. These results suggest that nose-drop application of monoclonal antibody could provide convenient and effective protection against RSV infection in human infants at risk of severe lower respiratory tract disease.

Human respiratory syncytial virus (RSV) is the leading cause of severe bronchiolitis and pneumonia in infants. RSV vaccine development has been stifled for the past 23 years because infants vaccinated with formalin-inactivated (FI) RSV have experienced exacerbated disease upon RSV infection. This exacerbated disease phenomenon is poorly understood, in part because of the lack of a primate model that exhibits a similar exacerbated disease state. Vaccination of African green monkeys with either FI RSV or a genetically engineered subunit vaccine termed FG glycoprotein reduced replication of challenge virus. However, only vaccination with FI RSV induced an enhanced pulmonary pathologic response to RSV infection. Pulmonary inflammatory scores in the FG glycoprotein-vaccinated monkeys were no greater than in monkeys vaccinated with adjuvant alone. This is the first demonstration of RSV vaccine-induced enhanced pathology in a primate and illustrates that a subunit vaccine has the potential of circumventing this exacerbated disease phenomenon.