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Biomedical subjects

B R Murphy

Publications and source records attributed to B R Murphy.

At least 145 records · Page 8Linked to original sources
PubMed1991

Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived.

The ability of recombinant vaccinia viruses that separately encoded 9 of the 10 known respiratory syncytial virus (RSV) proteins to induce resistance to RSV challenge was studied in BALB/c mice. Resistance was examined at two intervals following vaccination to examine early (day 9) as well as late (day 28) immunity. BALB/c mice were inoculated simultaneously by the intranasal and intraperitoneal routes with a recombinant vaccinia virus encoding one of the following RSV proteins: F, G, N, P, SH, M, 1B, 1C, or M2 (22K). A parainfluenza virus type 3 HN protein recombinant (Vac-HN) served as a negative control. One half of the mice were challenged with RSV intranasally on day 9, and the remaining animals were challenged on day 28 postvaccination. Mice previously immunized by infection with RSV, Vac-F, or Vac-G were completely or almost completely resistant to RSV challenge on both days. In contrast, immunization with Vac-HN, -P, -SH, -M, -1B, or -1C did not induce detectable resistance to RSV challenge. Mice previously infected with Vac-M2 or Vac-N exhibited significant but not complete resistance on day 9. However, in both cases resistance had largely waned by day 28 and was detectable only in mice immunized with Vac-M2. These results demonstrate that F and G proteins expressed by recombinant vaccinia viruses are the most effective RSV protective antigens. This study also suggests that RSV vaccines need only contain the F and G glycoproteins, because the immunity conferred by the other proteins is less effective and appears to wane rapidly with time.

Animals↗
PubMed1991

Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus.

The influenza A/Alaska/77 (H3N2) virus mutant 143-1 is temperature sensitive (ts) due to a spontaneous in-frame 36-nucleotide deletion in the nonstructural (NS) gene segment, which leads to a 12-amino-acid deletion in the NS1 protein. In addition, it has a small-plaque phenotype on MDCK cell monolayers. However, phenotypically revertant (i.e., ts+) viruses were isolated readily following replication of the 143-1 virus both in vitro and in vivo. In order to determine the genetic mechanism by which escape from the ts phenotype occurred, we performed segregational analysis and found that an intrasegmental suppressor mutation caused the loss of the ts phenotype. Nucleotide sequence analysis revealed the presence of an intragenic mutation in each of the ts+ phenotypic revertant viruses, involving a substitution of valine for alanine at amino acid 23 of the NS1 protein. This mutation resulted in acquisition of the ts+ phenotype and also in the large-plaque phenotype on MDCK cells, characteristic of the wild-type A/Alaska/77 parent virus. This amino acid substitution is predicted to generate an area of alpha helix in the secondary structure of the amino-terminal portion of the NS1 protein of the revertant viruses which may compensate for loss of an alpha-helical region due to the deletion of amino acids 66 to 77 in the NS1 protein of the 143-1 virus.

Amino Acid Sequence↗
PubMed1991

Thymic hyperplasia after chemotherapy: two case reports and a literature review.

The development of mediastinal masses in patients previously treated for cancer frequently indicates recurrent or metastatic disease. In this article we describe two patients previously treated for malignancies who had new or recurrent anterior mediastinal masses. These masses proved to be markedly enlarged thymuses due to thymic hyperplasia. The development of thymic hyperplasia after systemic chemotherapy is an uncommon but well known phenomenon. The management of such patients includes close observation or surgical intervention. Empiric antineoplastic therapy is not indicated.

Adolescent↗
PubMed1990

D-penicillamine metabolism: the role of transformation in blood plasma.

The transformation of D-penicillamine (D-pen) was studied in orally- and intravenously-dosed rats and in human plasma in vitro. In each case, low molecular weight (LMW) metabolites (previously identified as disulphides) and a mixed disulphide between D-pen and albumin (D-pen-protein) formed. The rates of D-pen elimination, other than through protein conjugation, were comparable in the rat groups to the rate of oxidation to LMW metabolites in vitro. The rates of transformation to D-pen-protein were also comparable in the in vitro preparations and in orally-treated rats. These qualitative and quantitative similarities suggest blood plasma may be an important site of transformation in vivo. Extracellular oxidation of D-pen may be linked to its antirheumatic action, either through reduction of oxygen species or through formation of D-pen-protein disulphides at surfaces of mononuclear leukocytes.

Administration, Oral↗
PubMed1990

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)

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Evaluation in chimpanzees of vaccinia virus recombinants that express the surface glycoproteins of human respiratory syncytial virus.

The immunogenicity and protective efficacy of recombinant vaccinia viruses that express the two major protective antigens of human respiratory syncytial virus (RSV), the F and G glycoproteins, were evaluated in chimpanzees. In previous studies in rodents and monkeys the F and G proteins expressed by the same recombinants were highly immunogenic and induced high levels of resistance to RSV replication following subsequent challenge. In contrast, in chimpanzees, a single intradermal immunization induced only moderate levels of F and G-specific serum antibodies as measured by an enzyme-linked immunosorbent assay, and these antibodies did not efficiently neutralize RSV infectivity in vitro. This poor antibody response in chimpanzees to the F and G glycoproteins occurred despite efficient replication of the vaccinia virus vector as evidenced by lesion size and serum antibody response to vaccinia virus. Upon intranasal RSV challenge, it was observed that prior immunization with the F and G recombinants effected only a marginal reduction in the magnitude and duration of RSV shedding from the nose and trachea and did not reduce illness. However, the RSV challenge induced a strong secondary antibody response, resulting in very high titres (greater than 8000 reciprocal mean titre) of serum neutralizing antibodies. The poor protective efficacy observed here is discussed with regard to the permissiveness of the chimpanzee to RSV replication, the general requirements for effective immunization against RSV, and the limitations of experimental animals for evaluating candidate RSV vaccines.

Animals↗
PubMed1990

Attenuation and immunogenicity in primates of vaccinia virus recombinants expressing human interleukin-2.

Vector-directed lymphokine expression represents a novel approach to the attenuation of live recombinant viruses which might be used as vaccines. Expression of interleukin-2 (IL-2) by recombinant vaccinia virus has been shown to significantly attenuate virus virulence in rodent species without diminishing immunogenicity. Skin lesion formation and immunogenicity of vaccinia/IL-2 recombinants in three species of primates was examined. IL-2 expression was associated with a 15-fold reduction in the area of induration after intradermal inoculation of recombinant viruses in patas monkeys. Wild type and a control vaccinia recombinant produced large (greater than 5000 mm2) skin ulcers in this species, but the IL-2 expressing recombinant produced no ulceration. Production of antibodies to vaccinia virus and to influenza A virus haemagglutinin expressed by recombinant vectors was examined in rhesus and squirrel monkeys. IL-2 expression accelerated the resolution of skin lesions in rhesus but not squirrel monkeys. Despite this, antibody production was equivalent in the presence or absence of IL-2. IL-2 expression can greatly reduce the skin lesions formed by live recombinant vaccinia vectors in primates, indicating significant attenuation, without reducing the immunogenicity of the vaccine.

Animals↗
PubMed1990

Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV 3-6 months after immunization.

Formalin-inactivated (FI) RSV, purified F glycoprotein in alum, and RSV infection (intranasal) were compared for their immunogenicity, efficacy, and ability to enhance pulmonary histopathology during RSV infection 3 and 6 months following immunization by the intramuscular route. Purified influenza virus in alum was used as a control immunogen. At 1 month following immunization with one dose of purified F glycoprotein (5 micrograms), cotton rats developed levels of F antibodies (ELISA) higher than the other groups, but these antibodies had the lowest level of neutralizing activity, Little increase in antibody titre was seen following a second dose of FI-RSV or purified F vaccine given at 1 month. Animals that received 5 micrograms F, 0.5 microgram F, or were almost completely resistant to pulmonary RSV infection following challenge at 3 months, but were susceptible by 6 months. Animals immunized with 5 micrograms of purified F glycoprotein developed alveolar and bronchiolar histopathology following RSV challenge at 3 or 6 months which was comparable to that of animals immunized with FI-RSV. These levels significantly exceeded those in animals previously immunized with influenza A virus vaccine which exhibited little histopathology. Animals previously infected with RSV also developed bronchiolar, but not alveolar, histopathology suggesting that the bronchiolar histopathology seen in RSV challenged cotton rats is a normal component of the immune resolution of RSV infection. These results suggest that the immune response of cotton rats to immunoaffinity purified F glycoprotein can result in enhanced bronchiolar and alveolar histopathology following RSV challenge. Thus, caution should be exercised in studies in humans using a purified F glycoprotein subunit vaccine.

Animals↗
PubMed1990

Evaluation of the infectivity, immunogenicity, and efficacy of live cold-adapted influenza B/Ann Arbor/1/86 reassortant virus vaccine in adult volunteers.

A cold-adapted (ca) influenza B reassortant that derived two genes encoding the hemagglutinin and neuraminidase from influenza B/Ann Arbor/1/86 wild-type virus and six internal RNA segments from ca influenza B/Ann Arbor/1/66 virus was evaluated in 66 adult volunteers having a serum hemagglutination inhibition antibody titer less than or equal to 1:8. The ca reassortant was attenuated and elicited the production of systemic and local antibodies; the 50% human infectious dose was 10(6.4) TCID50. Six weeks after vaccination, 12 unvaccinated volunteers and 13 recipients of ca virus (10(7.5) TCID50) were challenged experimentally with homologous wild-type influenza B virus. The ca vaccine completely protected against illness, and the magnitude of shedding was 50-fold less in vaccinees than in unimmunized controls, five of whom became ill. These findings indicate that the six internal RNA segments of the ca influenza B/Ann Arbor/66 donor virus confer desirable properties of a live virus vaccine to a reassortant derived from a virulent virus. Such reassortants may be suitable vaccines for healthy adults.

Adult↗
PubMed1990

Comparison of live attenuated cold-adapted and avian-human influenza A/Bethesda/85 (H3N2) reassortant virus vaccines in infants and children.

Randomized, placebo-controlled studies with 10(3)-10(7) 50% tissue-culture infectious dose (TCID50) of avian-human (ah) and cold-adapted (ca) influenza A/Bethesda/85 (H3N2) reassortant viruses were completed in 106 seronegative young children 6-48 months of age. Although the reassortants differed in six of eight RNA segments, they exhibited similar properties in level of attenuation, infectivity, immunogenicity, and efficacy. The 50% human infectious dose was 10(4.6) TCID50 for ah and 10(4.4) for ca vaccines. Both reassortants were satisfactorily attenuated with restricted replication and were no more reactogenic than placebo. The mean peak titer of virus shed was 10(1.5) (ah) to 10(2.0) (ca) TCID50/ml, and each of 37 isolates tested retained their characteristic vaccine phenotypes. Infection with ah or ca virus conferred immunity to experimental challenge with homologous virus. These findings indicate that both ah and ca influenza A/Bethesda/85 (H3N2) reassortants should be suitable vaccine candidates for use in healthy infants and young children.

Antibodies, Viral↗
PubMed1990

Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice.

The M2 protein of influenza A virus is expressed on the surfaces of infected cells, and a monoclonal antibody to this protein inhibits plaque enlargement of sensitive influenza A viruses without reducing plaque titer (S.L. Zebedee and R.A. Lamb, J. Virol. 62:2762-2772, 1988). In the current study, passively transferred monoclonal antibody to M2 reduced the level of replication of influenza A virus but not of influenza B virus in the lungs of mice. These experiments demonstrated that antibody to a protein conserved among influenza A virus subtypes inhibits virus growth in vivo.

Animals↗
PubMed1990

Antibody responses of humans and nonhuman primates to individual antigenic sites of the hemagglutinin-neuraminidase and fusion glycoproteins after primary infection or reinfection with parainfluenza type 3 virus.

An unusual feature of human parainfluenza virus type 3 (PIV3) is ita ability to cause reinfection with high efficiency. The antibody responses of 45 humans and 9 rhesus monkeys to primary infection or subsequent reinfection with PIV3 were examined to identify deficiencies in host immunologic responses that might contribute to the ability of the virus to cause reinfection with high frequency. Antibody responses in serum were tested by using neutralization and hemagglutination inhibition (HI) assays and a monoclonal antibody blocking immunoassay able to detect antibodies to epitopes within six antigenic sites on the PIV3 hemagglutinin-neuraminidase (HN) glycoprotein and eight antigenic sites on the fusion (F) protein. Primary infection of seronegative infants or children with PIV3 stimulated strong and rather uniform HI and neutralizing antibody responses. More than 90% of the individuals developed antibodies to four of the six HN antigenic sites (including three of the four neutralization sites), but the responses to F antigenic sites were of lesser magnitude and varied considerably from person to person. Young infants who possessed maternally derived antibodies in their sera developed lower levels and less frequent HI, neutralizing, and antigenic site-specific responses to the HN and F glycoproteins than did seronegative infants and children. In contrast, children reinfected with PIV3 developed even higher HI and neutralizing antibody responses than those observed during primary infection. Reinfection broadened the HN and F antigenic site-specific responses, but the latter remained relatively restricted. Adults possessed lower levels of HI, neutralizing, and antigenic site-specific antibodies in their sera than did children who had been reinfected, suggesting that these antibodies decay with time. Rhesus monkeys developed more vigorous primary and secondary antibody responses than did humans, but even in these highly responsive animals, response to the F glycoprotein was relatively restricted following primary infection. Bovine PIV3 induced a broader response to human PIV3 in monkeys than was anticipated on the basis of their known relatedness as defined by using monoclonal antibodies to human PIV3. These observations suggest that the restricted antibody responses to multiple antigenic sites on the F glycoprotein in young seronegative infants and children and the decreased responses to both the F and HN glycoproteins in young infants and children with maternally derived antibodies may play a role in the susceptibility of human infants and young children to reinfection with PIV3.

Adult↗
PubMed1989

The B allele of the NS gene of avian influenza viruses, but not the A allele, attenuates a human influenza A virus for squirrel monkeys.

The nonstructural (NS) genes of avian influenza A viruses have been divided into two groups on the basis of nucleotide sequence homology, which we have referred to here as alleles A and B. We sequenced the NS genes of eight additional avian influenza A viruses in order to define the differences between these two alleles more thoroughly. Four of the viruses had NS gene sequences which resembled that of A/FPV/Rostock/34 and belonged to allele A while the other four viruses had NS gene sequences more similar to that of A/Duck/Alberta/76 and belonged to allele B. There was approximately 90% sequence homology within alleles and 72% homology between alleles. As previously reported the NS genes of human influenza A viruses belong to allele A. We constructed single gene avian-human reassortant influenza A viruses containing an allele A or B NS gene segment from an avian influenza A virus and all other genes from a human influenza A virus and tested these reassortants for their ability to grow in the respiratory tract of a nonhuman primate. Reassortants containing an avian NS gene segment of allele B were significantly restricted in growth in the respiratory tract of squirrel monkeys while reassortants with an allele A NS gene segment were not. The divergent evolution of the B NS allele in birds may have resulted in gene products which do not function optimally in cooperation with genes from a human virus in viral replication in primate respiratory epithelium.

Alleles↗
PubMed1989

Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease.

A formalin-inactivated respiratory syncytial virus (RSV) vaccine tested 22 years ago failed to protect infant vaccinees against RSV infection or disease. Instead, lower respiratory tract disease was enhanced during subsequent infection by RSV. Enhancement of pulmonary pathology is also observed when cotton rats are immunized with formalin-inactivated RSV and subsequently infected with this virus. A major question that must be addressed for each new paramyxovirus vaccine is whether the immunogen possesses the capacity to potentiate disease. In the present study, we evaluated a newly developed purified F and G glycoprotein vaccine over a wide dosage range for immunogenicity, efficacy and capacity to potentiate pulmonary pathology in cotton rats. In addition, a formalin-inactivated RSV vaccine, which served as a positive control for enhancement of pulmonary pathology, was evaluated simultaneously. The results of these comparisons indicate that the purified F and G glycoprotein vaccine was highly immunogenic and was efficacious even in animals that developed low levels of serum-neutralizing antibodies. Furthermore, the F and G vaccine did not induce potentiation of pulmonary pathology. In contrast, formalin-inactivated RSV potentiated RSV pulmonary histopathology, but there was a sparing of potentiation at high and low doses. Both the formalin-inactivated RSV and purified F and G preparations induced a high level of serum antibodies capable of binding to purified F and G glycoproteins but both sets of antibodies had significantly reduced neutralizing activity. These results are encouraging because they suggest that purified paramyxovirus glycoproteins might be used safely as a vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Characterization of the M protein and nucleoprotein genes of an avian influenza A virus which are involved in host range restriction in monkeys.

A reassortant virus possessing RNA segment 7, which codes for the M1 and M2 proteins, of the avian influenza A/Mallard/New York/6750/78 (H2N2) virus and the other seven RNA segments of the human influenza A/Udorn/307/72 (H3N2) virus had been shown previously to be markedly restricted in replication in the respiratory tract of squirrel monkeys. In contrast, a reassortant possessing segment 7 of another avian influenza virus, A/Pintail/Alberta/119/79 (H4N6), and the seven other RNA segments from the A/Udorn/72 virus was not restricted. The nucleotide and deduced amino acid sequence of the RNA segment 7 of each virus was determined to identify the structural basis for the attenuation phenotype specified by RNA segment 7 of the A/Mallard/78 virus. Analysis of the deduced amino acid sequences revealed only two amino acid differences in the M1 protein and one difference in the M2 protein, suggesting that the attenuation phenotype of a reassortant virus possessing segment 7 of the A/Mallard/78 virus may be specified by one to three amino acids. Reassortant viruses possessing RNA segment 6, which codes for the nucleoprotein, of either avian influenza virus and the other seven RNA segments of a human influenza virus were also restricted in replication in squirrel monkeys. A comparison of the deduced amino acid sequences of the two avian nucleopeoteins demonstrated only three amino acid differences indicating that these two avian viruses possess NP genes that are highly related. The high degree of relatedness of both the NP and M proteins of these two avian viruses contrasts with their divergent surface antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗