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Previous studies have suggested that infant vaccinations may reduce the risk of subsequent childhood leukaemia. Vaccination histories were compared in 439 children (ages 0-14) diagnosed with acute lymphoblastic leukaemia (ALL) in nine Midwestern and Mid-Atlantic states (USA) between 1 January 1989 and 30 June 1993 and 439 controls selected by random-digit dialing and individually matched to cases on age, race and telephone exchange. Among matched pairs, similar proportions of cases and controls had received at least one dose of oral poliovirus (98%), diphtheria-tetanus-pertussis (97%), and measles-mumps-rubella (90%) vaccines. Only 47% of cases and 53% of controls had received any Haemophilus influenzae type b (Hib) vaccine (relative risk (RR) = 0.73; 95% confidence interval (CI) 0.50-1.06). Although similar proportions of cases (12%) and controls (11%) received the polysaccharide Hib vaccine (RR = 1.13; 95% CI 0.64-1.98), more controls (41%) than cases (35%) received the conjugate Hib vaccine (RR = 0.57; 95% CI 0.36-0.89). Although we found no relationship between most infant vaccinations and subsequent risk of childhood ALL, our findings suggest that infants receiving the conjugate Hib vaccine may be at reduced risk of subsequent childhood acute lymphoblastic leukemia. Further studies are needed to confirm this association and, if confirmed, to elucidate the underlying mechanism.
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.
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.
Respiratory viruses such as respiratory syncytial virus (RSV), the parainfluenza viruses (PIV), and the influenza viruses cause severe lower respiratory tract diseases in infants and children throughout the world. Experimental live attenuated vaccines for each of these viruses are being developed for intranasal administration in the first weeks or months of life. A variety of promising RSV, PIV-3, and influenza virus vaccine strains have been developed by classical biological methods, evaluated extensively in preclinical and clinical studies, and shown to be attenuated and genetically stable. The ongoing clinical evaluation of these vaccine candidates, coupled with recent major advances in the ability to develop genetically engineered viruses with specified mutations, may allow the rapid development of respiratory virus strains that possess ideal levels of replicative capacity and genetic stability in vivo. A major remaining obstacle to successful immunization of infants against respiratory virus associated disease may be the relatively poor immune response of very young infants to primary virus infection. This paper reviews the immune correlates of protection against disease caused by these viruses, immune responses of infants to naturally-acquired infection, and immune responses of infants to experimental infection with candidate vaccine viruses.
Two live-attenuated, cold-passaged (cp), temperature-sensitive (ts) candidate vaccines, designated cpts530/1009 and cpts248/955, were attenuated, genetically stable, and immunogenic in chimpanzees and were highly attenuated for human adults. In respiratory syncytial virus (RSV)-seropositive children, cpts530/1009 was more restricted in replication than cpts248/955. In seronegative children, 10(4) pfu of cpts248/955 was insufficiently attenuated, and a high titer of vaccine virus was shed (mean peak titer, 10(4.4) pfu/mL), whereas 10(4) pfu of cpts530/1009 was relatively attenuated and restricted in replication (mean peak titer, 10(2.0) pfu/mL). At a dose of 10(5) pfu, cpts530/1009 was immunogenic in seronegative children (geometric mean titer of RSV neutralizing antibodies, 1:724). Transmission of either vaccine to seronegative placebo recipients occurred at a frequency of 20%-25%. Of importance, vaccine viruses recovered from chimpanzees and humans were ts. In contrast to previous studies, this study indicates that live attenuated RSV vaccines that are immunogenic and phenotypically stable can be developed. Additional studies are being conducted to identify a live RSV vaccine that is slightly more attenuated and less transmissible than cpts530/1009.
Although a randomized clinical trial has yet to show a statistically significant improvement in the survival of patients receiving vaccine therapy for malignant melanoma, several studies have shown enhanced survival of patients developing an immune response to a melanoma vaccine. The knowledge and techniques of modern molecular biology and immunology suggest multiple strategies to augment this response. The challenge of immunotherapy research is to determine which combination of approaches leads to a favorable clinical response and how to monitor that response effectively. This review identifies components of a successful vaccine, discusses new ways to modulate and stimulate the immune system, and summarizes some of the more interesting clinical trials of melanoma vaccine immunotherapy.
cpts530, a candidate live-virus vaccine, is an attenuated strain of human respiratory syncytial virus (RSV). It was derived by subjecting a cold-passaged (cp) strain of RSV to a single round of chemical mutagenesis. cpts530 is a temperature-sensitive (ts) mutant that is attenuated in mice and chimpanzees, and its ts phenotype exhibits a high level of stability during replication in both species. In the present study, the complete nucleotide sequence of cpts530 RSV was determined. The five mutations known to be present in the parent cpRSV were retained in its cpts530 derivative, and one additional nucleotide change was identified at nucleotide (nt) 10060, which resulted in a phenylalanine-to-leucine change at amino acid 521 in the large polymerase (L) protein. To determine if this single amino acid substitution was indeed responsible for the ts phenotype of cpts530, it was introduced alone or in combination with the cp mutations into the full-length cDNA clone of the wild-type A2 RSV. Analysis of infectious viruses recovered from mutant cDNAs indicated that this single mutation specified complete restriction of plaque formation of recombinant cp530 in HEp-2 cell monolayer cultures at 40 degrees C, and the level of temperature sensitivity was not influenced by the presence of the five cpRSV mutations. These findings identify the phenylalanine-to-leucine change at amino acid 521 in the L protein as the mutation that specifies the ts phenotype of cpts530. Furthermore, these findings illustrate the feasibility of using the cDNA-based recovery system to analyze and construct defined attenuated vaccine viruses.
A cold-passaged (cp) temperature-sensitive (ts) mutant of human respiratory syncytial virus designated RSV cpts-248 was previously derived by random chemical mutagenesis of the non-ts mutant cp-RSV that possesses one or more host range mutations. We previously demonstrated in rodents and seronegative chimpanzees that the cpts-248 virus is more attenuated than cp-RSV and is more stable genetically than previously isolated RSV ts mutants. In the present study, we determined that the acquisition of the ts phenotype and the increased attenuation of the cpts-248 virus are associated with a single nucleotide substitution at nucleotide 10,989 that results in a change in the coding region (amino acid position 831) of the polymerase gene. The identification of this attenuating ts mutation is important because cpts-248 was used as the parent virus for the generation of a number of further attenuated mutants that are currently being evaluated as candidate vaccine strains in clinical trials in infants. Furthermore, technology now exists to rationally design new vaccine candidates by incorporating multiple attenuating mutations, such as the one identified here, into infectious viruses that are genetically stable and appropriately attenuated.
The complete nucleotide sequence of the RSV cpts-248/404 live attenuated vaccine candidate was determined from cloned cDNA and was compared to that of the RSV A2/HEK7 wild-type, cold-passaged cp-RSV, and cpts-248 virus, which constitute the series of progenitor viruses. RSV cpts-248/404 is more attenuated and more temperature sensitive (ts) (shut-off temperature 36 degrees) than its cpts-248 parent virus (shut-off temperature 38 degrees) and is currently being evaluated in phase I clinical trials in humans. Our ultimate goal is to identify the genetic basis for the host range attenuation phenotype exhibited by cp-RSV (i.e., efficient replication in tissue culture but decreased replication in chimpanzees and humans) and for the ts and attenuation phenotypes of its chemically mutagenized derivatives, cpts-248 and cpts-248/404. Compared with its cpts-248 parent, the cpts-248/404 virus possesses an amino acid change in the polymerase (L) protein and a single nucleotide substitution in the M2 gene start sequence. In total, the cpts-248/404 mutant differs from its wild-type RSV A2/HEK7 progenitor in seven amino acids [four in the polymerase (L) protein, two in the fusion (F) glycoprotein, and one in the (N) nucleoprotein] and one nucleotide difference in the M2 gene start sequence. Heterogeneity at nucleotide position 4 (G or C, negative sense, compared to G in the RSV A2/HEK7 progenitor) in the leader region of vRNA developed during passage of the cpts-248/404 in tissue culture. Biologically cloned derivatives of RSV cpts-248/404 virus that differed at position 4 possessed the same level of temperature sensitivity and exhibited the same level of replication in the upper and lower respiratory tract of mice, suggesting that heterogeneity at this position is not clinically relevant. The determination of the nucleotide sequence of the cpts-248/404 virus will allow evaluation of the stability of the eight mutations that are associated with the attenuation phenotype during vaccine production and following replication in humans.
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.