Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region.

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.

Evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy.

A live-attenuated, intranasal respiratory syncytial virus (RSV) candidate vaccine, cpts-248/404, was tested in phase 1 trials in 114 children, including 37 1-2-month-old infants-a target age for RSV vaccines. The cpts-248/404 vaccine was infectious at 104 and 105 plaque-forming units in RSV-naive children and was broadly immunogenic in children >6 months old. Serum and nasal antibody responses in 1-2 month olds were restricted to IgA, had a dominant response to RSV G protein, and had no increase in neutralizing activity. Nevertheless, there was restricted virus shedding on challenge with a second vaccine dose and preliminary evidence for protection from symptomatic disease on natural reexposure. The cpts-248/404 vaccine candidate did not cause fever or lower respiratory tract illness. In the youngest infants, however, cpts-248/404 was unacceptable because of upper respiratory tract congestion associated with peak virus recovery. A live attenuated RSV vaccine for the youngest infant will use cpts-248/404 modified by additional attenuating mutations.

Evaluation of the live attenuated cpts 248/404 RSV vaccine in combination with a subunit RSV vaccine (PFP-2) in healthy young and older adults.

The safety and immunogenicity of the live attenuated cold-passaged, temperature-sensitive (cpts) 248/404 respiratory syncytial virus (RSV) A2 and the RSV A2 purified F glycoprotein (PFP-2) vaccine candidates were evaluated in a placebo-controlled trial in 60 healthy young adults and 60 healthy elderly subjects using simultaneous and sequential (cpts 248/404 followed by PFP-2) vaccination schedules. Both vaccines were well tolerated. The cpts 248/404 vaccine was moderately infectious in both young and old volunteers, but was highly restricted in replication in those who were infected. After both vaccines, RSV neutralizing antibody (neut Ab) titers increased fourfold in 22% of young subjects and in 16% of elderly subjects. Of those with low levels of RSV neut Ab (titer <9), 10/12 (83% of) young subjects and six/eight (75% of) elderly subjects had a >/=four fold rise in neut Ab titer. Young and elderly subjects immunized simultaneously had similar serum IgG and IgA postimmunization titers to RSV F (IgG, 16.4 vs 16.2, IgA 11.6 vs 12. 5, respectively) as did those who were immunized sequentially (IgG 17.4 vs 17.0, IgA 13.0 vs 13.5). In both age groups, sequential immunization elicited higher postimmunization RSV F IgG and IgA titers than simultaneous immunization. Further studies that combine the PFP-2 subunit vaccine with a less attenuated RSV vaccine should be performed.

Rational design of live-attenuated recombinant vaccine virus for human respiratory syncytial virus by reverse genetics.

RSV is a major cause of pediatric respiratory tract disease worldwide, but a vaccine is not yet available. It is now possible to prepare live infectious RSV completely from cDNA. This provides a method for introducing defined mutations into infectious virus, making possible the rational design of a live-attenuated vaccine virus for intranasal administration. This is particularly important for RSV, for which achieving the appropriate balance between attenuation and immunogenicity by conventional methods has proven elusive. We took advantage of the existence of a panel of biologically derived vaccine candidate viruses that were incompletely attenuated but well characterized biologically. The mutations in these viruses were identified by sequence analysis and characterized by insertion into recombinant virus, thereby providing a menu of known attenuating mutations. These included a series of amino acid point mutations, mostly in the L polymerase, and a nucleotide substitution in a transcription gene-start signal, a cis-acting RNA element. The second source of mutations was from experimental mutational analysis of recombinant virus and involves deletion of the NS1, NS2, or SH gene. We have reconstructed a previously tested, biologically derived attenuated virus, cpts248/404, in recombinant form and are now proceeding to introduce additional mutations from the menu to achieve stepwise increases in attenuation. The ability to modify the attenuation phenotype incrementally in a directed manner should result in an appropriate vaccine virus.

Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines.

A number of antibodies generated during human respiratory syncytial virus (RSV) infection have been cloned by the phage library approach. Antibodies reactive with an immunodominant epitope on the F glycoprotein of this virus have a high affinity for affinity-purified F antigen. These antibodies, however, have a much lower affinity for mature F glycoprotein on the surface of infected cells and are nonneutralizing. In contrast, a potent neutralizing antibody has a high affinity for mature F protein but a much lower affinity for purified F protein or F protein in viral lysates. The data indicate that at least two F protein immunogens are produced during natural RSV infection: immature F, found in viral lysates, and mature F, found on infected cells or virions. Binding studies with polyclonal human immunoglobulin G suggest that the antibody responses to the two immunogens are of similar magnitudes. Competitive binding studies suggest that overlap between the responses is relatively limited. A mature envelope with an antigenic configuration different from that of the immature envelope has an evolutionary advantage in that the infecting virus is less subject to neutralization by the humoral response to the immature envelope that inevitably arises following lysis of infected cells. Subunit vaccines may be at a disadvantage because they most often resemble immature envelope molecules and ignore this aspect of viral evasion.

Isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (Fab RSVF2-5) that exhibits therapeutic efficacy in vivo.

A second human respiratory syncytial virus (RSV)-neutralizing monoclonal antibody was isolated and its binding site was identified. Fab F2-5 is a broadly reactive fusion (F) protein-specific recombinant Fab generated by antigen selection from a random combinatorial library displayed on the surface of filamentous phage. In an in vitro plaque-reduction test, the Fab RSVF2-5 neutralized the infectivity of a variety of field isolates representing viruses of both RSV subgroups A and B. The Fab recognized an antigenic determinant that differed from the only other human anti-F monoclonal antibody (RSV Fab 19) described thus far. A single dose of 4.0 mg of Fab RSVF2-5/kg of body weight administered by inhalation was sufficient to achieve a 2000-fold reduction in pulmonary virus titer in RSV-infected mice. The antigen-binding domain of Fab RSVF2-5 offers promise as part of a prophylactic regimen for RSV infection in humans.

Monoclonal antibody-resistant mutants selected with a respiratory syncytial virus-neutralizing human antibody fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein.

A recombinant human antibody fragment, designated RSV Fab 19, efficiently neutralizes respiratory syncytial virus (RSV). Here we report the results of our sequence analysis of antibody escape mutants that identified F glycoprotein amino acids critical for binding of human or murine RSV F-neutralizing antibodies.

Construction of four double gene substitution human x bovine rotavirus reassortant vaccine candidates: each bears two outer capsid human rotavirus genes, one encoding P serotype 1A and the other encoding G serotype 1, 2, 3, or 4 specificity.

Previously, four human x bovine rotavirus reassortant candidate vaccines, each of which derived ten genes from bovine rotavirus UK strain and only the outer capsid protein VP7-gene from human rotavirus strain D (G serotype 1), DS-1 (G serotype 2), P (G serotype 3), or ST3 (G serotype 4), were developed [Midthun et al., (1985): Journal of Virology 53:949-954; (1986): Journal of Clinical Microbiology 24:822-826]. Such human x bovine reassortant vaccines should theoretically provide antigenic coverage for the four epidemiologically most important VP7(G) serotypes 1, 2, 3, and 4. In an attempt to increase the antigenicity of VP7-based human x animal reassortant rotavirus vaccines which derive a single VP7-encoding gene from the human strain and the remaining ten genes from the animal strain, we generated double gene substitution reassortants. This was done by incorporating another protective antigen (VP4) of an epidemiologically important human rotavirus by crossing human rotavirus Wa strain (P serotype 1A), with each of the human x bovine single VP7-gene substitution rotavirus reassortants. In this way four separate double gene substitution rotavirus reassortants were generated. Each of these reassortants bears the VP4-encoding gene from human rotavirus Wa strain, the VP7-encoding gene from human rotavirus strain D, DS-1, P, or ST3, and the remaining nine genes from bovine rotavirus strain UK. The safety, antigenicity, and protective efficacy of individual components as well as combinations of strains are currently under evaluation.

Efficacy of a quadrivalent rhesus rotavirus-based human rotavirus vaccine aimed at preventing severe rotavirus diarrhea in infants and young children.

The most extensively explored strategy for rotavirus vaccination has been the Jennerian approach, which uses an antigenically related rotavirus strain from an animal host as the immunogen to induce protection against the 4 epidemiologically important group A rotavirus VP7 serotypes. Because this approach has shown limited efficacy, a modified Jennerian approach was developed with the goal of achieving broader antigenic coverage. Four VP7 serotypes were incorporated into a quadrivalent vaccine comprised of three rhesus-human rotavirus reassortants, each with 10 rhesus rotavirus genes and 1 human rotavirus gene that encodes VP7 serotype 1, 2, or 4 specificity; the rhesus rotavirus itself provides coverage for VP7 serotype 3. This approach appears quite promising for preventing severe rotavirus diarrhea, including those episodes that lead to dehydration. Additional strategies under development stress the role not only of human rotavirus VP7 but also of human rotavirus VP4, the other outer capsid protein that also induces neutralizing antibodies.

Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys.

The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3' noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5' boundary of the 3' noncoding region and whose 3' boundary did not extend beyond the last 113 nt of the 3' end were viable. With the exception of mutant 3'd 303-183, which contained a deletion of nt 303 to 183 from the 3' terminus, deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK2 cells than parental wild-type virus. Significantly, mutant 3'd 303-183 grew to a high titer and was least restricted in growth. Mutant 3'd 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3'd 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization. The immunogenicity of these 3' DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK2 cells. One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

Live subgroup B respiratory syncytial virus vaccines that are attenuated, genetically stable, and immunogenic in rodents and nonhuman primates.

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.

Jennerian and modified Jennerian approach to vaccination against rotavirus diarrhea using a quadrivalent rhesus rotavirus (RRV) and human-RRV reassortant vaccine.

Rotaviruses are the single most important cause of severe diarrhea of infants and young children world-wide. Deaths from rotavirus diarrhea occur infrequently in developed countries; however, in developing countries, rotaviruses are estimated to cause over 870000 deaths in the under five-year age group. There is, therefore, a vital need for a vaccine to prevent severe rotavirus diarrhea in infants and young children. The most extensively evaluated strategy for rotavirus vaccination has been the "Jennerian" approach in which an antigenically related rotavirus strain from an animal host (bovine or simian [rhesus monkey]) is used as the immunogen to induce protection against the four epidemiologically important group A human rotavirus serotypes. These orally administered vaccines were safe and immunogenic but had only limited success because serotype-specific immunity was not induced consistently in the under six-month age group. Therefore, a modified "Jennerian" approach was adopted with the goal of attaining broader antigenic coverage. In this approach four serotypes are combined to form a quadrivalent vaccine comprised of (i) rhesus rotavirus (RRV) which provides coverage for VP7 serotype 3, and (ii) three human-RRV reassortants each with ten RRV genes and a single human rotavirus gene that encodes VP7 serotype 1, 2, or 4 specificity. This modified "Jennerian" approach appears to be quite promising in preventing severe diarrhea in field trials. However, if this approach fails to yield an optimal level of protection consistently, additional modified "Jennerian" strategic, are under development that consider not only human rotavirus VP7 but also human rotavirus VP4, the other outer capsid protein. In addition, a non-"Jennerian" approach includes the development of cold-adapted human rotavirus strains or cold-adapted human rotavirus reassortants as vaccine candidates.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Cold-passaged, temperature-sensitive mutants of human respiratory syncytial virus (RSV) are highly attenuated, immunogenic, and protective in seronegative chimpanzees, even when RSV antibodies are infused shortly before immunization.

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)

Isolation and characterization of a highly attenuated respiratory syncytial virus (RSV) vaccine candidate by mutagenesis of the incompletely attenuated RSV A2 ts-1 NG-1 mutant virus.

Ts-1, a temperature sensitive (ts) mutant of RSV, was previously derived from RSV A2 virus by mutagenesis with 5-fluorouracil (5-FU). Ts-1 was attenuated for adult volunteers and seropositive children but retained a low level of virulence in seronegative infant vaccinees as indicated by the occurrence of upper respiratory tract disease. Ts-1 NG-1, a more defective derivative of ts-1, was produced by mutagenesis of ts-1 with nitrosoguanidine. However, ts-1 NG-1 still retained a low level of virulence for the upper respiratory tract and showed some genetic instability in chimpanzees. With renewed interest in the goal of developing a live, attenuated RSV vaccine, we have now attempted to further attenuate ts-1 NG-1 by mutagenesis with 5-FU and 5-azacytidine. Four mutants that are phenotypically different from the ts-1 NG-1 parental virus were identified. Each of the four mutants was more restricted in replication in BALB/c mice compared with the ts-1 NG-1 parental virus. One of the ts-1 NG-1 derivatives, termed A-20-4, which showed the lowest (35 degrees C) in vitro shutoff temperature and which was also completely restricted in replication in BALB/c mice, was selected for further evaluation in seronegative chimpanzees. A-20-4 did not cause rhinorrhea in chimpanzees but induced detectable titers of serum RSV neutralizing antibodies in 2 of 4 chimpanzees. Apparent complete protection to subsequent challenge with wild-type RSV was observed in each of the four chimpanzees previously immunized with A-20-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection of cold-adapted mutants of human rotaviruses that exhibit various degrees of growth restriction in vitro.

Group A human rotavirus strains D, Wa, DS-1, and P were originally recovered from children with diarrhea. In an attempt to attenuate virulent, wild-type human rotaviruses of major epidemiological importance for use in a live oral vaccine, two reference rotavirus strains, D and DS-1, and two laboratory-generated reassortants, Wa x DS-1 and Wa x P, were subjected to cold adaptation. Collectively, these viruses provide antigenic coverage for both of the clinically important rotavirus VP4 antigens and three of the four important rotavirus VP7 antigens. Mutants of each of these rotaviruses were selected during successive serial passage in primary African green monkey kidney cells at progressively lower suboptimal temperatures (30, 28, and 26 degrees C). The genotype of each mutant appeared to be indistinguishable from that of its wild-type, parental virus. The mutants recovered after 10 serial passages at 30 degrees C exhibited both temperature sensitivity of plaque formation (i.e., a ts phenotype) and the ability to form plaques efficiently at suboptimal temperature (i.e., a cold adaptation [ca] phenotype), in contrast to parental wild-type rotavirus. The succeeding set of 10 serial passages at 28 degrees C selected mutants that exhibited an increased degree of cold adaptation, and three of the mutants exhibited an associated increase in temperature sensitivity. Finally, in the case of three of the strains, the third successive serial passage series, which was performed at 26 degrees C, selected for mutants with an even greater degree of cold adaptation than the previous series and was associated with greater temperature sensitivity in one instance. It appeared that each of the viruses sustained a minimum of four to five mutations during the total selection procedure. The ultimate identification of candidate vaccine viruses that exhibit the desired level of attenuation, immunogenicity, and protective efficacy needed for immunoprophylaxis will require evaluation of these mutants in susceptible humans.

Isolation and characterization of a chimpanzee monoclonal antibody to the G glycoprotein of human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory tract disease in infants and young children. In this study a hybridoma line secreting a chimpanzee monoclonal antibody that neutralizes RSV was isolated. Two chimpanzees were immunized with recombinant vaccinia viruses that express the RSV F or G surface glycoprotein and 1 month later were infected intranasally with the wild-type RSV strain A2. Peripheral blood lymphocytes obtained from the animals were transformed with Epstein-Barr virus, and lymphoblastoid cell lines that secreted anti-RSV antibodies were identified by an RSV antigen-binding enzyme-linked immunosorbent assay. Supernatants from RSV antibody-secreting lymphoblastoid cell lines were tested for in vitro virus neutralization before being fused to the heteromyeloma cell GLI-H7. A chimpanzee antibody [immunoglobulin G3(lambda) subclass] produced from a hybridoma line designated E1.4/2 was shown to bind to the RSV G glycoprotein and neutralize a panel of subgroup A viruses, but not subgroup B viruses, at low (nanomolar) concentrations. Mice passively immunized with this antibody were partially resistant to RSV strain A2 challenge. The usefulness of such antibodies in immunoprophylaxis and immunotherapy of RSV infection is discussed.

A further attenuated derivative of a cold-passaged temperature-sensitive mutant of human respiratory syncytial virus retains immunogenicity and protective efficacy against wild-type challenge in seronegative chimpanzees.

A cold-passage (cp), temperature-sensitive (ts) RSV mutant designated RSV cpts-248 (shut-off temperature 38 degrees C), which possesses host-range mutations acquired during 52 passages at low temperature in bovine tissue culture and a ts phenotype introduced by subsequent chemical mutagenesis, was found previously to be attenuated, immunogenic, and protective against wild-type challenge in seronegative chimpanzees. We sought to introduce additional attenuating mutations such as small-plaque (sp) and ts mutations into RSV cpts-248 by chemical mutagenesis with 5-fluorouracil with the intent of obtaining cpts-248 derivatives that are more attenuated in mice or chimpanzees and that are more genetically stable following replication in vivo. Ten mutants of RSV cpts-248 which had acquired a sp phenotype or a second ts mutation were generated by chemical mutagenesis. Five cpts-248 derivatives which had acquired mutations that specified a 36 degrees C shut-off temperature for plaque formation and one which had acquired only a sp phenotype were more restricted in replication in Balb/c mice than the cpts-248 parental strain. One mutant, designated RSV cpts-248/404 (shut-off temperature 36 degrees C), was 100 times more restricted in replication in the nasal turbinates of mice and 100 times more restricted in the nasopharynx of seronegative chimpanzees than its cpts-248 parent. The cpts-248/404 mutant was completely restricted in replication in the lower respiratory tract of chimpanzees even following direct intratracheal administration. The ts phenotype of the cpts-248/404 mutant was stable during replication in vivo in mice and chimpanzees.(ABSTRACT TRUNCATED AT 250 WORDS)