A recombinant chimeric La Crosse virus expressing the surface glycoproteins of Jamestown Canyon virus is immunogenic and protective against challenge with either parental virus in mice or monkeys.

La Crosse virus (LACV) and Jamestown Canyon virus (JCV), family Bunyaviridae, are mosquito-borne viruses that are endemic in North America and recognized as etiologic agents of encephalitis in humans. Both viruses belong to the California encephalitis virus serogroup, which causes 70 to 100 cases of encephalitis a year. As a first step in creating live attenuated viral vaccine candidates for this serogroup, we have generated a recombinant LACV expressing the attachment/fusion glycoproteins of JCV. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. For the M segment, the open reading frame (ORF) of LACV is replaced with that derived from JCV and is flanked by the untranslated regions of LACV. The resulting chimeric virus retained the same robust growth kinetics in tissue culture as observed for either parent virus, and the virus remains highly infectious and immunogenic in mice. Although both LACV and JCV are highly neurovirulent in 21 day-old mice, with 50% lethal dose (LD50) values of 0.1 and 0.5 log10 PFU, respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 10³ PFU. Parenteral vaccination of mice with 10¹ or 10³ PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and induced neutralizing antibodies to JCV, LACV, and TAHV. When vaccinated monkeys were challenged with JCV, they were protected against the development of viremia. Generation of highly attenuated yet immunogenic chimeric bunyaviruses could be an efficient general method for development of vaccines effective against these pathogenic viruses.

Factors modifying host resistance to viral infection. 3. Effect of whole body x-irradiation on experimental encephalomyocarditis virus infection in mice.

The resistance of mice to encephalomyocarditis (EMC) virus was markedly decreased by prior exposure to whole body X-irradiation. In contrast to non-irradiated controls, the course of EMC virus infection in X-irradiated animals was characterized by (a) an enhanced mortality, (b) shortening of the incubation period, (c) higher levels of virus in the blood during the viremic phase and persistence of the viremia until death, (d) failure to develop detectable serum levels of neutralizing antibody, and (e) the earlier appearance and higher levels of virus in brain and heart tissue. The level of interferon in the serum during the course of infection was similar in both groups. The administration of relatively small quantities of anti-EMC virus neutralizing antibody to X-irradiated mice during the early phases of the infection with EMC virus restored their resistance to levels comparable to nonirradiated animals. An alteration of local organ defense mechanisms in the central nervous system could not be demonstrated. It is proposed that (a) the inability of the X-irradiated animal to elaborate specific neutralizing antibody was a critical determinant in their failure to clear the viremia, (b) this increase in the level and duration of the viremic phase resulted in the exposure of target organs to a greater inoculum of virus, and (c) the enhanced mortality observed in irradiated mice reflected this greater target organ involvement. The experimental model presented, therefore, suggests that the immunologic response is a critical determinant of host resistance during this primary systemic virus infection.

The effects of feeding history and environment on condition, body composition and growth of bluegills Lepomis macrochirus.

Initial relative mass (W(R), low v. high) and energetic trajectory in time (starved v. fed) were experimentally manipulated in bluegill Lepomis macrochirus. Fed fish starting at low W(R) grew more and gained more W(R) than fed fish starting at high W(R). Similarly, starved fish starting at high W(R) lost more mass and W(R) than did starved fish starting at low W(R). Temporal changes in other variables did not consistently match that of W(R), but, by the end of the experiment, proximate composition showed a high correlation to W(R). Regression slopes of W(R) on proximate composition increased with time in the laboratory. Differences between wild and laboratory fish appeared to result from relaxation of environmental stress. When excess resources are available such that L. macrochirus grow, condition indices will increase, but individual response will depend on initial values and thus past environmental experience.

Reassortant virus derived from avian and human influenza A viruses is attenuated and immunogenic in monkeys.

An influenza A reassortant virus that contained the hemagglutinin and neuraminidase genes of a virulent human virus, A/Udorn/72 (H3N2), and the six other influenza A virus genome segments from an avirulent avian virus, A/Mallard/New York/6750/78 (H2N2), was evaluated for its level of replication is squirrel monkeys and hamsters. In monkeys, the reassortant virus was as attenuated and as restricted in its level of replication in the upper and lower respiratory tract as its avian influenza virus parent. Nonetheless, infection with the reassortant induced significant resistant to challenge with virulent human influenza virus. In hamsters, the reassortant virus replicated to a level intermediate between that of its parents. These findings suggest that the nonsurface antigen genes of the avian parental virus are the primary determinants of restriction of replication of the reassortant virus in monkeys. Attenuation of the reassortant virus for primates is achieved by inefficient functioning of the avian influenza genes in primate cells, while antigenic specificity of the human influenza virus is provided by the neuraminidase and hemagglutinin genes derived from the human virus. This approach could lead to the development of a live influenza A virus vaccine that is attenuated for man if the avian influenza genes are similarly restricted in human cells.

Transduction of folate receptor cDNA into cervical carcinoma cells using recombinant adeno-associated virions delays cell proliferation in vitro and in vivo.

Although folate receptors (FRs) mediate folate uptake into cells, the independent role of FRs in cell proliferation remains unclear. We tested the hypothesis that transduction of FR cDNA in sense or antisense orientation using recombinant adeno-associated virus modulated FR expression and altered proliferation of cervical carcinoma cells (which constitutively overexpress FR genes). We determined that the integration of recombinant adeno-associated virions was not site specific. When compared with untransduced cells, sense and antisense FR cDNA-transduced cells exhibited an increase and decrease in FR mRNA and FR expression on the cell surface, respectively. However, when compared with antisense FR cDNA-transduced and untransduced cells, sense FR cDNA-transduced cells exhibited statistically significant (a) increased in total FRs, (b) smaller colonies, (c) lowered cell proliferation in vitro, and (d) less tumor volume with dramatic prolongation of tumor doubling times (225.6 h vs. 96 h) after transplantation into nude mice. Finally, (f) using single cell-derived transduced clones, an inverse relationship between cell proliferation and FR expression was established (r = 0.90, P < 0.001). Thus, transduction of sense/antisense FR cDNA into cervical carcinoma cells modulated expression of FRs and had an impact on cell proliferation in vitro and in vivo.

Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine.

BACKGROUND: Studies in animal models have shown that systemic immunization with a papillomavirus virus-like particle (VLP) vaccine composed of L1, a major structural viral protein, can confer protection against subsequent experimental challenge with the homologous virus. Here we report results of a double-blind, placebo-controlled, dose-escalation trial to evaluate the safety and immunogenicity of a human papillomavirus (HPV) type 16 (HPV16) L1 VLP vaccine in healthy adults. METHODS: Volunteers were given intramuscular injections with placebo or with 10- or 50-microg doses of HPV16 L1 VLP vaccine given without adjuvant or with alum or MF59 as adjuvants at 0, 1, and 4 months. All vaccine recipients were monitored for clinical signs and symptoms for 7 days after each inoculation. Immune responses were measured by an HPV16 L1 VLP-based enzyme-linked immunosorbent assay (ELISA) and by an HPV16 pseudovirion neutralization assay. The antibody titers were given as the reciprocals of the highest dilution showing positive reactivity in each assay. All statistical tests were two-sided. RESULTS: The prevaccination geometric mean ELISA titer for six seropositive individuals was 202 (range, 40--640). All vaccine formulations were well tolerated, and all subjects receiving vaccine seroconverted. Serum antibody responses at 1 month after the third injection were dose dependent in recipients of vaccine without adjuvant or with MF59 but were similar at both doses when alum was the adjuvant. With the higher dose, the geometric means of serum ELISA antibody titers (95% confidence intervals) to purified VLP 1 month after the third injection were as follows: 10,240 (1499 to 69 938) without adjuvant, 10,240 (1114 to 94 145) with MF59, and 2190 (838 to 5723) with alum. Responses of subjects within each group were similar. Neutralizing and ELISA antibody titers were highly correlated (Spearman correlation =.85), confirming that ELISA titers are valid proxies for neutralizing antibodies. CONCLUSIONS: The HPV16 L1 VLP vaccine is well tolerated and is highly immunogenic even without adjuvant, with the majority of the recipients achieving serum antibody titers that were approximately 40-fold higher than what is observed in natural infection.

Surgical salvage of chemorefractory germ cell tumors.

PURPOSE: Patients with disseminated germ cell tumors who relapse after salvage chemotherapy, or who progress during cisplatin-based therapy, have chemorefractory disease and a very poor prognosis. A subset of these patients will have chemorefractory but resectable disease. We have therefore evaluated the role of salvage surgery in this patient population. PATIENTS AND METHODS: We performed a retrospective review of all patients with disseminated germ cell tumors who were felt to have chemorefractory disease and underwent salvage surgery from 1977 to 1990 at Indiana University. All patients had elevated serum markers or other signs of progressive carcinoma. A total of 48 patients underwent surgery (33 retroperitoneal lymph node dissections [RPLNDs], six thoracotomies, three thoracoabdominal resections, and multiple asynchronous procedures in six patients). RESULTS: Thirty-eight of 48 patients (79%) were rendered grossly free of disease and 29 (60%) obtained a serologic remission. Ten patients (21%) remain continuously disease-free with no postoperative treatment with a median follow-up of 46 months (range, 31 to 89). Six additional patients who relapsed after salvage surgery are currently disease-free with further treatment (four with repeat surgery and two with high-dose chemotherapy and autologous bone marrow transplantation [ABMT]). CONCLUSION: Selected patients with chemorefractory but resectable germ cell tumors have definite potential for cure with salvage surgery.

The live attenuated subgroup B respiratory syncytial virus vaccine candidate RSV 2B33F is attenuated and immunogenic in chimpanzees, but exhibits partial loss of the ts phenotype following replication in vivo.

The cold-adapted (ca), temperature-sensitive (ts) respiratory syncytial virus (RSV) subgroup B vaccine candidate, designated RSV 2B33F, was found previously to be restricted in replication, immunogenic, and protective against wild-type (wt) virus challenge in rodents and African green monkeys. We sought to investigate the level of attenuation, immunogenicity and genetic stability of this vaccine candidate in seronegative chimpanzees. The 2B33F vaccine candidate was attenuated in chimpanzees and manifested a ten- and 1000-fold restriction in replication in the upper and lower respiratory tracts respectively, compared with its wt RSV 2B parent virus. Despite this attenuation, chimpanzees immunized with RSV 2B33F were completely resistant to respiratory tract disease and virus replication upon challenge with wt virus. The ts phenotype of the RSV 2B33F mutant exhibited some alteration during replication in vivo in three of four chimpanzees tested. Virus present in nasopharyngeal swab or tracheal lavage secretions of these three chimpanzees was biologically cloned by plaque passage in Vero cells at permissive temperature. The plaque progeny retained the ts phenotype, but uniformly produced plaques at 39 and 40 degrees C to a level intermediate between that of the 2B33F input virus and the 2B wt parent virus, indicating that partial loss of the level of temperature sensitivity occurred following replication in vivo. The implications of these findings for RSV vaccine development are discussed.

The complete nucleotide sequence of the JS strain of human parainfluenza virus type 3: comparison with the Wash/47885/57 prototype strain.

The nucleotide sequence of the JS strain of human parainfluenza virus type 3 (PIV3) was determined from a series of 14 overlapping cDNA clones and was compared to that of the previously sequenced prototype PIV3 strain, Wash/47885/57 (Galinski, 1991). Overall, there were 630 (4%) nucleotide differences between the two viruses. 15462 nucleotides comprised the JS genome in contrast to 15463 which constituted the genome of the prototype virus. This was accounted for by a single nucleotide deletion in the 5' non-coding region of the JS phosphoprotein gene. Four nucleotide substitutions were found in the leader region at the 3' end of the viral genome at positions 24, 28, 42 and 45, whereas no differences were found in the 44 base trailer region. All of the transcription start and stop signals and intergenic sequences were conserved between the two viruses with the exception of the transcription stop signal of the matrix (M) gene where there was a nucleotide transposition between bases 7 and 8. A comparison of all of the nucleotide differences in the 3' and 5' non-coding regions of each gene showed a variability of 9.8% and 10.5%, respectively. The 3' non-coding regions of the nucleocapsid (NP) and M genes were completely conserved in contrast to the polymerase (L) gene in which 25% of the nucleotides were different. Differences were observed in the 5' non-coding regions of each gene and ranged from 5.9% for the hemagglutinin neuraminidase (HN) gene to 14.6% for the M gene. An analysis of the amino acid differences in each open reading frame revealed that of all the genes, the coding region of the M gene was the most highly conserved (1.1% amino acid variability), while the phosphoprotein (P) gene was the most variable (5.8% amino acid variability). As these two viruses are wild type strains, these differences in nucleotide and amino acid sequence are compatible with efficient replication in vivo.

The attenuation phenotype conferred by the M gene of the influenza A/Ann Arbor/6/60 cold-adapted virus (H2N2) on the A/Korea/82 (H3N2) reassortant virus results from a gene constellation effect.

A single gene reassortant (SGR) virus that derived its M gene from the attenuated influenza A/Ann Arbor/6/60 cold-adapted (CA) donor virus and the remaining genes from the A/Korea/82 (H3N2) wild type (WT) virus (designated A/Korea/82 CA M-SGR) was previously shown to be attenuated in mice, hamsters, ferrets, and humans. The attenuation (ATT) phenotype of this SGR virus could result directly from an altered function of the mutant M gene product of the A/Ann Arbor/6/60 CA virus, which differs from the M gene of the A/Ann Arbor/6/60 WT virus at only one amino acid or, indirectly from a gene constellation effect in which ATT results from an inefficient interaction between the products of the M gene of the A/Ann Arbor/6/60 virus and other genes of the A/Korea/82 virus. Several lines of evidence from the present study are consistent with our interpretation that the ATT phenotype of the A/Korea/82 CA M-SGR results from a gene constellation effect. First, the A/Korea/82 CA M-SGR and an A/Korea/82 SGR containing the A/Ann Arbor/6/60 WT M gene were each restricted in replication in the upper and lower respiratory tract of mice compared with the A/Korea/82 WT virus. Second, an A/Udorn/72 CA M-SGR containing the M gene from the A/Ann Arbor/6/60 CA donor virus in a background of other genes derived from the A/Udorn/72 (H3N2) WT virus was not attenuated in the respiratory tract of mice. These data suggest that the change in the amino acid sequence of the M gene product from the A/Ann Arbor/6/60 WT to CA virus is not responsible for the ATT phenotype of the A/Korea/82 CA M-SGR. In addition, evidence of the genetic instability of the A/Korea/82 CA M-SGR is presented, specifically, an extragenic mutation that results in loss of the ATT phenotype. The implications of these findings for the ATT phenotype of the live attenuated reassortant viruses derived from the A/Ann Arbor/6/60 CA donor virus are discussed.

Are there alternative avian influenza viruses for generation of stable attenuated avian-human influenza A reassortant viruses?

The present study evaluated gull influenza A viruses as donors of attenuating genes for the production of live, attenuated influenza A H1N1 and H3N2 avian-human (ah) reassortant viruses for use as vaccines to prevent disease due to influenza A viruses in humans. The previously evaluated duck influenza A viruses were abandoned as donors of attenuating avian influenza virus genes because clinical evaluation of H1N1 and H3N2 ah reassortant virus vaccines derived from duck viruses documented residual virulence of H1N1 reassortants for seronegative infants and young children. Gull influenza A viruses occupy an independent ecologic niche and are rarely isolated from species other than gulls. The possibility of using gull influenza A viruses as donors of internal gene segments in ah reassortant viruses was evaluated in the present study using three different gull viruses and three human influenza A viruses. Gull-human H3N2 reassortant influenza A viruses with the desired 6-2 genotype (six internal avian influenza virus genes and the two human influenza virus surface glycoprotein genes) were readily generated and were found to be attenuated for squirrel monkeys and chimpanzees. However, ah reassortant viruses with gull and human influenza A H1N1 genes were difficult to generate, and reassortants that had the desired genotype of six gull virus genes with human influenza A H1 and N1 genes were not isolated despite repeated attempts. The gull PB2, NP and NS genes were not present in any of the gull-human H1N1 reassortants generated. The under-representation of these three gene segments suggests that reassortants bearing one or more of these three gene segments might have reduced viability indicative of a functional incompatibility in their gene products. The difficulties encountered in the generation of a 6-2 gull-human H1N1 reassortant virus are sufficient to conclude that the gull influenza A viruses tested would not be useful as donors of sets of six internal genes to attenuate human influenza A viruses. This study also identifies influenza virus gene segments that appear to be incompatible for generation of reassortants. Elucidation of the molecular basis of this restriction may provide information on intergenic interactions involved in virion assembly or packaging.

Detection of respiratory syncytial virus (RSV) infected cells by in situ hybridization in the lungs of cotton rats immunized with formalin-inactivated virus or purified RSV F and G glycoprotein subunit vaccine and challenged with RSV.

The replication of RSV in unimmunized cotton rats was evaluated by quantitating the amount of infectious virus in the lung and the number of RSV infected cells in a histopathological section of lung by in situ hybridization. RSV infected cells were detected only in alveoli and bronchioles and constituted only a small minority of the cell population. The temporal patterns of rise to the peak number of infected cells (day 4) and the peak titer of infectious virus (day 3) were similar. The clearance of both infected cells and infectious virus was nearly complete by day 7. In animals previously immunized with purified RSV glycoproteins or formalin-inactivated RSV there also was a good correlation between the number of infected cells detected by in situ hybridization and the amount of infectious virus recovered. It was previously demonstrated that cotton rats immunized with formalin-inactivated vaccine developed enhanced pulmonary histopathology following challenge with RSV. In such animals, there was approximately a 90% reduction in the number of infected cells compared to control unimmunized, RSV-challenged animals. Formalin-inactivated RSV vaccine-enhanced lung histopathology developed despite the effective elimination of virus and virus-infected cells suggesting that the enhanced pathology is the result of an exaggeration of normal immune mechanisms involved in clearance of virus infection, an aberrant immune response during infection, or both.

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype.

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

The complete nucleotide sequence of two cold-adapted, temperature-sensitive attenuated mutant vaccine viruses (cp12 and cp45) derived from the JS strain of human parainfluenza virus type 3 (PIV3).

Two cold-passaged mutant vaccine viruses (cp12 and cp45) derived from the JS wild-type (wt) strain of human parainfluenza virus type 3 (PIV3) have been sequenced. These mutant viruses display the cold-adapted (ca), temperature-sensitive (ts), and attenuation (att) phenotypes. Sequence data indicate that both cp12 and cp45 sustained nucleotide substitutions during cold passage and subsequent cloning. Fifteen nucleotide changes were present in cp12 and 18 in cp45. Of these changes, some were present in the sequence of the prototype wt strain (Wash/47885/57) or were non-coding changes present in the open reading frames (ORFs). These were considered unlikely to be of significance in contributing to phenotypic differences between the mutants and the JS wt. There were nine remaining changes in cp12 and eight in cp45 that would most likely contribute to their phenotypes. For cp12, two were non-coding changes in regulatory regions, one in the 3' genome leader and one in the NP gene transcription start signal. The remaining seven changes resulted in amino acid substitutions in NP, F, HN, and L. For cp45, two mutations were in a non-coding regulatory region, the 3' genome leader. The remaining six changes resulted in amino acid substitutions in F, HN, and L. Only one amino acid substitution was conserved between cp12 and cp45 (a valine to alanine change at position 384 of the HN gene). These results should prove useful in the future in understanding the genetic basis of attenuation of the cold-passaged PIV3 candidate vaccine viruses.

An update on approaches to the development of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) vaccines.

RSV and PIV3 are responsible for about 30% of severe viral respiratory tract disease leading to hospitalization of infants and children. For this reason, there is a need to develop vaccines effective against these viruses. Since these viruses cause severe disease in early infancy, vaccines must be effective in the presence of maternal antibody. Currently, several strategies for immunization against these viruses are being explored including peptide vaccines, subunit vaccines, vectored vaccines (e.g., vaccinia-RSV or adenovirus-RSV recombinants), and live attenuated virus vaccines. The current status of these approaches is reviewed. In addition, the immunologic basis for the disease potentiation seen in vaccinees immunized with formalin-inactivated RSV during subsequent RSV infection is reviewed. The efficacy of immunization in the presence of maternal antibody is discussed. Much progress for a RSV and PIV3 vaccine has been made and successful immunization against each of these pathogens should be achieved within this decade.

Cold-passaged human parainfluenza type 3 viruses contain ts and non-ts mutations leading to attenuation in rhesus monkeys.

Cold-passaged (CP) mutants derived from the JS strain of wild type wt parainfluenza type 3 virus (PIV3) are being evaluated as candidate live virus vaccines. The wt virus was serially passaged 45 times at low temperature and mutant clones with the cold-adapted (CA), temperature-sensitive (ts), and attenuation (ATT) phenotypes were selected following passage levels 12, 18 and 45 (cp12, cp18, and cp45). The cp45 virus was more ts than the cp12 or cp18 mutants, although all 3 mutant viruses were clearly attenuated in rhesus monkeys compared to wild type virus. The mean peak titers of the cp12 and cp18 viruses administered by the intratracheal route were at least 6000-fold lower than JSwt in both the upper and lower respiratory tracts. The cp45 virus was not recovered from monkeys administered virus by the i.t. route alone; however, when the cp45 virus was administered by the intranasal route, it replicated in the upper respiratory tract to a level comparable to that of the cp12 and cp18 viruses, but continued to be markedly restricted in the lower respiratory tract. These data indicate that the cp12 and cp18 viruses contain predominantly non-ts attenuating mutations whereas the cp45 mutant has both non-ts and ts attenuating mutations. Each of the CP mutants induced a high level of resistance to wild type virus challenge. Also, the ATT phenotype of the cp12 and cp18 viruses as measured in rhesus monkeys was stable after replication in chimpanzees or humans, respectively, although the ts phenotype was not. Based on its greater level of temperature sensitivity in vitro and its greater degree of attenuation in rhesus monkeys, the cp45 virus appears to be the most promising vaccine candidate for humans.

Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses.

Vaccines against parainfluenza (PIV) and respiratory syncytial viruses (RSV) that are currently being developed include both live and subunit vaccines. Candidate live PIV vaccines that have been found to be attenuated and efficacious in rodents or primate models are (1) cold-adapted, temperature-sensitive mutants of PIV-type 3 that have been serially passaged at low temperature (20 degrees C) in simian kidney tissue culture; (2) protease-activation mutants (PIV-1-Sendai), which have mutations that decrease the cleavability of their F glycoprotein by host cell protease; (3) an animal virus, bovine PIV-3 virus, which is antigenically related to the human PIV-3 virus, and (4) vaccinia recombinant viruses bearing RSV or PIV-3 glycoproteins. Subunit RSV and PIV-3 viruses are being produced and evaluated as immunogens. A major concern with these vaccines is the possibility of disease potentiation following virus infection as occurred previously with formalin-inactivated measles and RSV vaccines. Studies indicate that PIV-3 and RSV glycoprotein vaccines are immunogenic and efficacious in animals but insufficient data exist to estimate their capacity to potentiate disease. However, since a cotton rat model is available to detect potentiated disease resulting from infection of cotton rats previously immunized with formalin-inactivated RSV vaccine, it is now possible to systematically evaluate new vaccines in experimental animals for disease potentiation before studies are initiated in humans. It is likely within the next several years that one or more of these PIV or RSV vaccines will be tested in humans for safety and immunogenicity.

Evaluation of a live attenuated bovine parainfluenza type 3 vaccine in two- to six-month-old infants.

BACKGROUND: A safe and effective parainfluenza type 3 (PIV-3) virus vaccine is needed to prevent serious PIV-3-associated illness in infants younger than 6 months of age. In previous studies a live bovine PIV-3 (BPIV-3) vaccine, which was developed to prevent human PIV-3 (HPIV-3) disease, was shown to be safe, infectious, immunogenic and phenotypically stable in 6- to 36-month-old infants and children. METHODS: The safety, infectivity and immunogenicity of a single dose of the BPIV-3 vaccine was evaluated in a randomized, placebo-controlled, double blinded trial in 19 infants 2 to 5.9 months of age and in 11 additional 6- to 36-month-old subjects. RESULTS: The BPIV-3 vaccine was well-tolerated in both age groups and infected 92% of those younger than 6 months and 89% of those older than 6 months of age. Serum hemagglutination-inhibition (HAI) antibody responses to HPIV-3 and to BPIV-3, respectively, were detected in 42 and 67% of the younger infants, compared with 70 and 85% of the older subjects. In the younger infants we analyzed the rate of antibody response by titer of maternally acquired antibodies; low titer was defined as a preimmunization serum HAI titer < 1:8 and high titer was defined as a preimmunization serum HAI titer > or = 1:8. Young infants with a low titer of maternally acquired antibodies were significantly more likely to respond to the BPIV-3 vaccine that those with a high titer (89% vs. none for serum HAI response to BPIV-3; P = 0.02, Fisher's exact test). CONCLUSIONS: This study demonstrated that the BPIV-3 vaccine was safe and infectious in infants younger than 6 months of age and was also immunogenic in the majority of these young infants. Additional studies are needed to determine whether two or more doses will enhance the immunogenicity of the BPIV-3 vaccine in young infants and to assess its safety and immunogenicity when given simultaneously with routine childhood immunizations.

Safety and immunogenicity of a cold-adapted influenza A (H1N1) reassortant virus vaccine administered to infants less than six months of age.

A safe and effective influenza vaccine is needed to prevent serious influenza illness in infants younger than 6 months of age. The purpose of this study was to determine whether two doses of the cold-adapted (ca) influenza A reassortant vaccine would be safe and immunogenic in this age group. In the first part of this study, infants received two doses of 10(5) or 10(6) 50% tissue culture-infectious dose (TCID50) of the ca influenza vaccine separately from routine immunizations. In the second part of this study two 10(6) TCID50 doses of the ca influenza vaccine were given with routine immunizations at 2 and 4 or 2 and 6 months of age. The ca influenza vaccine was well-tolerated by participants in both parts of this study. Two doses of the ca influenza vaccine were immunogenic in infants who received them separately from routine immunizations; 83% of vaccinees developed protective titers of serum hemagglutination-inhibition (HAI) antibody. In contrast, when the ca vaccine was administered with routine immunizations, protective HAI antibody titers were induced in only 20% of those immunized at 2 and 4 months of age and 50% of those immunized at 2 and 6 months of age. There were no statistically significant associations between HAI antibody response to ca influenza vaccination and dose schedule, presence of passively acquired maternal HAI antibody, ethnic group or breast-feeding status. Young age at the time of first immunization, however, appeared to correlate with decreased response to the hemagglutinin antigen of the influenza A virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced infectivity of cold-adapted influenza A H1N1 viruses in the elderly: correlation with serum and local antibodies.

OBJECTIVE: To compare young and elderly adults in terms of their immune responses and rates of infection following intranasal vaccination with a live attenuated influenza virus. DESIGN: Time series, comparing outcomes in young and elderly convenience sample. METHOD: Retrospective laboratory analysis of serum and nasal wash specimens collected during prior studies in which young or elderly volunteers had been inoculated with cold-adapted influenza A/Kawasaki/86 (H1N1) reassortant virus. SETTING: Johns Hopkins Center for Immunization Research. PARTICIPANTS: Healthy young and elderly adults with pre-vaccination serum hemagglutination inhibition (HAI) antibody titers less than or equal to 1:8. OUTCOME MEASUREMENTS: Antibody responses in serum and nasal washes. MAIN RESULTS: The proportion of vaccinees who developed any serum or local antibody response was higher in young compared with elderly subjects (20/20 vs 5/14, P less than 0.0005). Resistance to infection with cold-adapted virus correlated with pre-vaccination levels of serum immunoglobulin G (IgG), serum IgA, and nasal wash IgA antibody to whole virus antigen. Age was highly correlated with a lack of response to vaccine by simple regression, but not when data were adjusted for pre-existing antibody levels. CONCLUSIONS: Cold-adapted reassortant influenza A H1N1 viruses achieve lower rates of infection in elderly than young adults, primarily due to age-related differences in preexisting levels of immunity which may not be reflected by HAI titer.