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The recombinant dengue virus type-4 vaccine candidate 2AA30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3'-untranslated region of the viral genome. A clinical trial to evaluate the safety and immunogenicity of a single dose of 2Adelta30 was conducted with 20 adult human volunteers. The vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. Viremia was detectable in 14 volunteers at a mean level of 1.6 log10 plaque-forming units/ml of serum, although all 20 volunteers seroconverted with a seven-fold or greater increase in serum neutralizing antibody titer on day 28 post-vaccination (mean titer = 1:580). A mild, asymptomatic, macular rash developed in 10 volunteers, and a transient elevation in the serum level of alanine aminotransferase was noted in five volunteers. The low level of reactogenicity and high degree of immunogenicity of this vaccine candidate warrant its further evaluation and its use to create chimeric vaccine viruses expressing the structural genes of dengue virus types 1, 2, and 3.

Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create a wt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. The wt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants.

A phenylalanine to leucine mutation at position 521 in the L polymerase of cpts530, a live-attenuated respiratory syncytial virus (RSV) cold-passaged (cp), temperature-sensitive (ts) candidate vaccine, specifies the ts and attenuation (att) phenotypes. Sequence alignment of this region in the L proteins of several distantly related paramyxoviruses revealed that this phenylalanine is conserved. Using reverse genetics, the analogous phenylalanine at position 456 in the L protein of wild-type PIV3 was mutagenized to leucine (F456L). The resulting virus, designated r456(L), was ts (40 degrees C shut-off temperature of plaque formation), and its replication in the upper, but not the lower, respiratory tract of hamsters was 10-fold reduced compared with that of the recombinant wild-type PIV3 (rwt). Thus the phenylalanine to leucine mutation specified a similar level of temperature sensitivity and attenuation in two distantly related paramyxoviruses. We next sought to determine whether the addition of this mutation to the L protein of two rPIV3 candidate vaccine viruses, one bearing the three cp45 ts missense mutations in the L protein (rcp45(L)) and the other bearing all 15 cp45 mutations (rcp45), would further attenuate the viruses in vivo. Each rcp45 derivative to which the F456L mutation was added exhibited an increased level of temperature sensitivity. Furthermore rcp45(L)-456 and rcp45-456 were 100- to 1000-fold more restricted in replication in hamsters than their rcp45(L) and rcp45 parents. Despite the high level of restriction of replication in hamsters, immunization with rcp45-456 induced a moderate level of resistance to replication of PIV3 challenge virus. In contrast to the highly restricted replication observed in hamsters, rcp45-456 was only fivefold more restricted in the respiratory tract of chimpanzees than rcp45 and induced a comparable, moderate to high level of PIV3-specific serum antibodies. rcp45 and rcp45-456 viruses isolated from chimpanzees throughout the 2-week course of replication maintained the level of temperature sensitivity of their respective input viruses, illustrating their phenotypic stability. Thus the acquisition of the F456L mutation by the cp45 virus resulted in a small, incremental increase in its level of attenuation, indicating its possible usefulness in the fine tuning of the level of attenuation of the cp45 vaccine candidate. The ability to transfer mutations identified in heterologous paramyxoviruses, which in this case represent different subfamilies, greatly enhances our ability to rapidly develop novel parainfluenza virus candidate vaccines., (Copyright 1999 Academic Press.)

Two parainfluenza virus type 3 (PIV3) vaccine candidates-cp45, a live attenuated derivative of the JS wild type (wt), and a replication-defective vaccinia virus recombinant expressing the hemagglutinin-neuraminidase or fusion glycoprotein of human PIV3 (modified vaccinia virus Ankara [MVA]/PIV3 recombinants)-were evaluated in rhesus monkeys to determine whether successful immunization could be achieved in the presence of passively transferred PIV3 antibodies. The cp45 virus, administered intranasally (in) and intratracheally (it) in the presence of high levels of PIV3 antibodies, replicated efficiently in the nasopharynx and protected against challenge with wt human PIV3. The MVA recombinants, administered in, it, and intramuscularly in the absence of passive antibody, conferred protection against replication of PIV3 wt challenge virus, but this was largely abrogated when immunization occurred in the presence of passive antibodies. Because immunization for the prevention of HPIV3 disease must occur in early infancy when maternal antibodies are present, the live attenuated cp45 virus continues to be a promising vaccine candidate for this age group.

Respiratory syncytial virus (RSV) cpts530/1030 is an attenuated, temperature-sensitive subgroup A vaccine candidate derived previously from cold-passaged RSV (cpRSV) by two sequential rounds of chemical mutagenesis and biological selection. Here, cpts530/1030 was shown to be highly attenuated in the upper and lower respiratory tracts of seronegative chimpanzees. However, evaluation in seropositive children showed that it retains sufficient replicative capacity and virulence to preclude its direct use as a live attenuated vaccine. Nucleotide sequence analysis of the genome of cpts530/1030 showed that it had acquired two nucleotide substitutions (compared to its cpts530 parent), both of which were in the L gene: a silent mutation at nucleotide position 8821 (amino acid 108) and a missense mutation at nucleotide position 12458 resulting in a tyrosine-to-asparagine change at amino acid 1321, herein referred to as the 1030 mutation. It also contained the previously identified 530 missense mutation at nucleotide 10060 in the L gene. The genetic basis of attenuation of cpts530/1030 was defined by the introduction of the 530 and 1030 mutations into a cDNA clone of cpRSV, from which recombinant RSV was derived and analyzed to determine the contribution of each mutation to the temperature sensitivity (ts) and attenuation (att) phenotypes of cpts530/1030. The 530 mutation, derived from cpts530, was previously shown to be responsible for the ts and att phenotypes of that virus. In the present study, the 1030 mutation was shown to be responsible for the increased temperature sensitivity of cpts530/1030. In addition, the 1030 mutation was shown to be responsible for the increased level of attenuation of cpts530/1030 in the upper and lower respiratory tracts of mice. The 530 and 1030 mutations were additive in their effects on the ts and att phenotypes. It was possible to introduce the 1030 mutation, but not the 530 mutation, into an attenuated vaccine candidate with residual reactogenicity in very young infants, namely, cpts248/404, by use of reverse genetics. The inability to introduce the 530 mutation into the cpts248/404 virus was shown to be due to its incompatibility with the 248 missense mutation at the level of L protein function. The resulting rA2cp248/404/1030 mutant virus was more temperature sensitive and more attenuated than the cpts248/404 parent virus, making it a promising new RSV vaccine candidate created by use of reverse genetics to improve upon an existing vaccine virus.

Optimal immunization of neonates against disease caused by respiratory syncytial virus (RSV) probably will require multiple doses of a vaccine containing viruses of both subgroups A and B. Live subgroup B RSV mutants were generated containing multiple attenuating mutations, ts (temperature-sensitive) and non-ts (host range), that were introduced by prolonged passage in cell culture or by chemical mutagenesis. The cold-passaged (cp)-52 mutant was restricted in replication compared to wild type virus in rodents and nonhuman primates. In addition, the attenuation phenotype of cp-52 was stable after prolonged replication in immunosuppressed rodents. One or two ts mutations were then introduced into the cp-52 mutant to generate additional candidate vaccine strains that were more attenuated in vivo than the cp-52 parental virus. Tests in humans are being done to determine if one or more of the RSV B-1 mutants exhibit a satisfactory balance between attenuation and immunogenicity.

Three model systems were used to demonstrate the immunogenicity of highly attenuated and replication-defective recombinant MVA. (1) Intramuscular inoculation of MVA-IN-Fha/np induced humoral and cell-mediated immune responses in mice and protectively immunized them against a lethal respiratory challenge with influenza virus. Intranasal vaccination was also protective, although higher doses were needed. (2) In rhesus macaques, an immunization scheme involving intramuscular injections of MVA-SIVenv/gag/pol greatly reduced the severity of disease caused by an SIV challenge. (3) In a murine cancer model, immunization with MVA-beta gal prevented the establishment of tumor metastases and even prolonged life in animals with established tumors. These results, together with previous data on the safety of MVA in humans, suggest the potential usefulness of recombinant MVA for prophylactic vaccination and therapeutic treatment of infectious diseases and cancer.

A cold-passaged (cp) temperature-sensitive (ts) RSV mutant, designated RSV cpts-530, which possesses host-range mutations acquired during 52 passages at low temperature in bovine tissue culture and one or more ts mutations induced by chemical mutagenesis (shut-off temperature 39 degrees C) was found previously to be tenfold restricted in its replication in mice as compared to wild-type virus and stable genetically in nude mice. In the current study, we introduced additional attenuating mutations, such as small-plaque (sp) or ts mutations, into cpts-530 by chemical mutagenesis with 5-fluorouracil, with the intent of obtaining derivatives of cpts-530 that were more attenuated in mice or chimpanzees and that were more stable genetically following replication in vivo. Fourteen mutants of RSV cpts-530 which had acquired an additional ts mutation were identified and found to be more restricted in replication in BALB/c mice than the cpts-530 parental strain. One mutant, designated cpts-530/1009 (shut-off temperature 36 degrees C), was 30 times more restricted in replication in the nasal turbinates of mice and threefold more restricted in the nasopharynx of seronegative chimpanzees than its cpts-530 parent. Like its parent, this mutant was highly restricted (30,000-fold) in replication in the lower respiratory tract of chimpanzees even following direct intratracheal inoculation. The cpts-530 and cpts-530/1009 mutants exhibited a high level of stability of the ts phenotype during replication in chimpanzees.(ABSTRACT TRUNCATED AT 250 WORDS)

Simian immunodeficiency virus (SIV) infection of macaques is a useful and relevant model for evaluating candidate human immunodeficiency virus (HIV) vaccines. One important feature of this model is that SIV vaccines can be evaluated for their ability to prevent infection as well as to prevent or delay the onset of AIDS. In the present study, a group of macaques was vaccinated with whole inactivated SIV and challenged with peripheral blood mononuclear cells from an SIV-infected macaque. This challenge represented a rigorous and realistic test of the immunization protocol. All macaques became infected after challenge; however, immunized animals survived significantly longer (P < .03) than naive controls. These data suggest that similar vaccines administered to humans at risk for HIV-1 infection might delay or prevent AIDS even if the vaccine failed to prevent infection.