Biomedical subjects

B R Murphy

Publications and source records attributed to B R Murphy.

At least 91 records · Page 5Linked to original sources

PubMed1995

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)

Animals↗

PubMed1995

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)

Animals↗

PubMed1995

Human immune responses to influenza virus vaccines administered by systemic or mucosal routes.

Healthy adult volunteers were immunized by parenteral or oral routes with trivalent inactivated influenza vaccine (A/Chile/1/83 (H1N1), A/Mississippi/1/85 (H3N2), and B/Ann Arbor/1/86), or intranasally with live attenuated, cold-adapted influenza type A/Texas/1/85 (H1N1) reassortant virus. In all volunteers, cells spontaneously secreting IgA, IgG or IgM antibodies specific to influenza virus were detected in peripheral blood on days 6-13 after immunization, and specific IgA, IgG and IgM antibodies to influenza vaccine were measured in sera and external secretions (saliva and nasal lavage). Following systemic immunization, a raise in specific antibodies of all isotypes was observed in sera beginning on day 13. Although small variations in IgA and IgM antibodies in saliva and nasal lavages were detected, antigen-specific IgG significantly increased between days 13 and 27. Intranasal administration of attenuated virus induced IgA and IgG antibodies in serum as well as in secretions. Serum antibodies were not substantially influenced by oral immunization, only a small increase in all isotypes was observed in volunteers' sera 21 days after ingestion of vaccine. However, in secretions, antigen-specific IgA and IgG responses were detected one week after immunization and reached a peak response on day 20. These studies show that different routes of immunization can be effective for the induction of specific antibodies, and support the concept of the common mucosal immune system in humans by demonstrating that the oral or intranasal administration of antigen-induced specific antibodies of IgA isotype in external secretions, preceded by the transient appearance in peripheral blood of specific antibody-producing cells.

Administration, Intranasal↗

PubMed1995

A live human parainfluenza type 3 virus vaccine is attenuated and immunogenic in healthy infants and children.

The safety, infectivity, immunogenicity, and phenotypic stability of the cold-passaged (cp) candidate vaccine cp-45, a cold-adapted (ca), temperature-sensitive (ts) mutant of the JS strain of human parainfluenza virus type 3 (HPIV-3), was evaluated in 114 children 6 months to 10 years old in a randomized, placebo-controlled, double-blind trial. The cp-45 vaccine was well tolerated when given intranasally to parainfluenza virus type 3 (PIV-3)-seropositive and -seronegative children. With 10(4) or 10(5) TCID50 of cp-45 vaccine, 86% of seronegative vaccines were infected, 83% of whom shed virus at a mean peak titer of 10(22) pfu/mL. Virus present in respiratory specimens retained the ts phenotype, and each of 86 PIV-3 isolates tested retained both the ca and ts phenotypes. One dose of 10(5) TCID50 of vaccine induced a serum hemagglutination-inhibiting antibody response in 81% of vaccinees; the geometric mean titer was 1:32. These studies indicate that the cp-45 HPIV-3 vaccine is satisfactorily attenuated, infectious, immunogenic, and phenotypically stable and merits further evaluation in infants and young children.

Antibodies, Viral↗

PubMed1995

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)

Age Factors↗

PubMed1995

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine.

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.

Animals↗

PubMed1995

Cytotoxic T cells specific for a single peptide on the M2 protein of respiratory syncytial virus are the sole mediators of resistance induced by immunization with M2 encoded by a recombinant vaccinia virus.

We have studied the immunobiology of respiratory syncytial virus (RSV), a major cause of respiratory tract morbidity in children. As part of these studies, it was previously found that immunization of BALB/c (H-2d) mice with a recombinant vaccinia virus (rVV) which encoded the M2 protein of RSV provided complete protection against infection with RSV. This protection was transient and associated with M2-specific CD8+ T-cell (TCD8+) responses. In this study, we used two approaches to demonstrate that expression of an H-2Kd-restricted nonameric peptide (Ser Tyr Ile Gly Ser Ile Asn Asn Ile) corresponding to M2 residues 82 to 90 is necessary and sufficient to induce protective TCD8+ responses. First, infection of mice with an rVV which encoded the peptide M2Met82-90 induced levels of primary pulmonary TCD8+ and resistance to RSV challenge equivalent to that induced by infection with an rVV which expressed the complete M2 protein. Second, elimination of peptide binding to Kd by the replacement of Tyr with Arg at amino acid position 83 of the full-length protein completely abrogated the ability of an rVV-expressing full-length M2 to induce either M2-specific TCD8+ responses or resistance to RSV infection. These findings demonstrate that the M2(82-90) peptide is the sole determinant of immunity induced in BALB/c mice by the M2 protein and that a remarkably high level of transient resistance to infection with pulmonary virus is associated with TCD8+ responses to a single determinant.

Animals↗

PubMed1995

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.

Animals↗

PubMed1994

Recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fab is effective therapeutically when introduced directly into the lungs of RSV-infected mice.

Previously, recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fabs were generated by antigen selection from random combinatorial libraries displayed at the tip of filamentous phage. Two such Fabs, which exhibited high binding affinity for RSV F glycoprotein (a major protective antigen), were evaluated for therapeutic efficacy in infected mice just before or at the time of peak virus replication in the lungs. Fab 19, which neutralized RSV infectivity with high efficiency in tissue culture, was effective therapeutically when delivered directly into the lungs by intranasal instillation under anesthesia. In contrast, RSV Fab 126, which failed to neutralize virus in cell culture, did not exhibit a therapeutic effect under these conditions. The amount of Fab 19 required to effect a 5000- to 12,000-fold reduction in titer of RSV in the lungs within 24 hr was rather small. In four separate experiments, a single instillation of 12.9-50 micrograms of RSV Fab 19 was sufficient to achieve such a reduction in pulmonary virus in a 25g mouse. The use of Fabs instead of the whole immunoglobulin molecules from which they are derived reduced the protein content of a therapeutic dose. This is important because the protein load that can be delivered effectively into the lungs is limited. The therapeutic effect of a single treatment with Fab 19 was not sustained, so that a rebound in pulmonary virus titer occurred on the 2nd day after treatment. This rebound in pulmonary RSV titer could be prevented by treating infected mice with a single dose of Fab 19 daily for 3 days. These observations suggest that human monoclonal Fabs grown in Escherichia coli may prove useful in the treatment of serious RSV disease as well as diseases caused by other viruses where replication in vivo is limited primarily to the lumenal lining of the respiratory tract.

Administration, Intranasal↗

PubMed1994

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.

Adult↗

PubMed1994

Satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis.

A cold-passaged RSV mutant, designated cp-RSV, which acquired host range mutations during 52 passages at low temperature in bovine tissue culture, was completely attenuated for seropositive adults and children but retained the capacity to cause upper respiratory disease in seronegative infants. We sought to introduce additional attenuating mutations, such as temperature-sensitive (ts) and small-plaque (sp) mutations, into the cp-RSV mutant, which is a ts+ virus, in order to generate a mutant which would be satisfactorily attenuated in seronegative infants and young children. Nine mutants of cp-RSV, which had acquired either the ts or small-plaque sp phenotype, were generated by chemical mutagenesis with 5-fluorouracil. The two ts mutants with the lowest in vitro shut-off temperature, namely the cpts-248 (38 degrees C) and cpts-530 (39 degrees C) mutants, were the most restricted of the nine cp-RSV mutant progeny tested for efficiency of replication in Balb/c mice. In seronegative chimpanzees, the cpts-248 mutant replicated fourfold less efficiently in the nasopharynx and caused significantly less rhinorrhoea than its cp-RSV parent. The cpts-248 mutant virus, like its cp-RSV parent, was 1000-fold restricted in replication in the trachea compared with wild-type RSV. Previously, another candidate RSV live attenuated vaccine strain, a mutant designated ts-1, exhibited some instability of its ts phenotype following replication in susceptible humans or chimpanzees. Hence, we sought cp-RSV ts progeny that exhibited a greater degree of stability of the ts phenotype than the prototype ts-1 mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

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)

Animals↗

PubMed1994

A phase II trial of vinblastine plus dipyridamole in advanced renal cell carcinoma. A Hoosier Oncology Group Study.

One potential explanation for why renal cell carcinoma is usually poorly responsive to chemotherapy is intrinsic multidrug resistance. Dipyridamole (DP) is one of several agents known to bind to P-glycoprotein and potentially reverse multidrug resistance in vitro, and dosages needed to obtain appropriate levels in vivo appear to be well tolerated. The current study was undertaken to evaluate the combination of vinblastine (VLB) and DP in patients with advanced renal cell carcinoma and no prior chemotherapy. From August 1989 through December 1989, 15 patients with inoperable recurrent or metastatic renal cell carcinoma were treated with VLB 0.2 mg/kg (i.v. slow push) and concurrently received DP75 mg p.o. q.i.d. starting 48 hours before and continuing 48 hours after VLB administration. Courses were repeated every 3 weeks for a maximum of 8 weeks, or until disease progression or undue toxicities ensued. The predominant toxicities seen were mild neurotoxicity and leukopenia. Only 1 patient had grade IV leukopenia, and no lethal toxicities occurred. No objective responses were seen; 2 patients had stable disease for 29 and 30+ months. The median survival was 9 months (range: 2.5-30+). We conclude that at the dose and schedule used in this study, the combination of VLB and DP may be administered with acceptable toxicities, but is ineffective in the treatment of advanced renal cell carcinoma.

Adolescent↗

PubMed1994

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.

Animals↗

PubMed1994

Evaluation of the genetic stability of the temperature-sensitive PB2 gene mutation of the influenza A/Ann Arbor/6/60 cold-adapted vaccine virus.

A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-type virus is a temperature-sensitive (ts) virus with an in vitro shutoff temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at elevated temperatures in Madin-Darby canine kidney cells to generate ts+ phenotypic revertant viruses. Four ts+ phenotypically revertant viruses were derived independently, and each possessed a shutoff temperature for replication in vitro of > 40 degrees C. Each of the four phenotypically revertant viruses replicated efficiently in the upper and lower respiratory tracts of mice and hamsters, unlike the PB2 single-gene reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts+ revertants with wild-type virus yielded ts progeny in high frequency, indicating that the loss of ts phenotype was due to a suppressor mutation which was mapped to the PA gene in each of the four independently derived ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicted amino acid changes in the PA proteins of the revertant viruses. These studies suggest that single amino acid changes at aa 245 (Glu-Lys) or 347 (Asp-Asn) of the PA protein can completely suppress the ts and attenuation phenotypes specified by the Asp-Ser mutation at aa 265 of the PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.

Amino Acid Sequence↗

PubMed1994

Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.

The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.

Amino Acid Sequence↗

PubMed1994

Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10.

In previous studies, children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency than did controls during subsequent natural RSV infection. In earlier efforts to develop an animal model for this phenomenon, extensive pulmonary histopathology developed in FI-RSV-immunized cotton rats and mice subsequently challenged with RSV. In mice, depletion of CD4+ T cells at the time of RSV challenge completely abrogated this histopathology. Furthermore, the predominant cytokine mRNA present in lungs of FI-RSV-immunized mice during subsequent infection with RSV was that characteristically secreted by Th2 T cells, namely interleukin-4 (IL-4). In the present studies, we sought to determine the relative contributions of gamma interferon (IFN-gamma), IL-2, IL-4, and IL-10 to the lymphocytic infiltration into the lungs observed following RSV challenge of mice previously immunized with FI-RSV. Mice previously immunized with FI-RSV or infected with RSV were depleted of IFN-gamma, IL-2, IL-4, or IL-10 immediately before RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels was quantified. The phenomenon of pulmonary-histopathology potentiation by FI-RSV was reproduced in the present study, thereby allowing us to investigate the effect of cytokine depletion on the process. Simultaneous depletion of both IL-4 and IL-10 completely abrogated pulmonary histopathology in FI-RSV-immunized mice. Depletion of IL-4 alone significantly reduced bronchiolar, though not perivascular, histopathology. Depletion of IL-10 alone had no effect. Depletion of IFN-gamma, IL-2, or both together had no effect on the observed histopathology. These data indicate that FI-RSV immunization primes for a Th2-, IL-4-, and IL-10-dependent inflammatory response to subsequent RSV infection. It is possible that this process played a role in enhanced disease observed in infants and children immunized with FI-RSV.

Animals↗

PubMed1993

Beta 2-microglobulin-deficient mice can be protected against influenza A infection by vaccination with vaccinia-influenza recombinants expressing hemagglutinin and neuraminidase.

Immunity to viral infections includes both antibody and T cell components. The contributions of humoral and cell-mediated immune responses vary depending on virus and host factors. We have used an in vivo challenge system to examine protective immunity to influenza A(H1N1) virus infection in immunocompetent B6 (H-2b) mice, and in H-2b mice homozygous for disruption of the gene for beta 2-microglobulin, termed beta 2 mu(-/-) mice. The latter mice do not express conventional MHC class I complexes on cell surfaces and lack CD8+ class I-restricted T cells. Ten vaccinia virus recombinants, each expressing 1 of the 10 proteins of influenza virus, were used to immunize the mice. Normal mice were protected against challenge with influenza virus by vaccination with HA-VAC and NA-VAC, but not by any of the vaccinia vectors expressing one of the eight other influenza virus proteins nor by a mixture of all eight of the latter vectors. Similar results were observed in mice of H-2d or H-2k MHC haplotypes. The beta 2 mu(-/-) mice were also protected by immunization with HA-VAC and NA-VAC, demonstrating that classical CD8+ CTL responses were not required for protection. Depletion of CD4+ T cells in either normal or beta 2 mu(-/-) mice at the time of challenge had little or no effect on protection induced by HA-VAC or NA-VAC, suggesting that preformed antibody is the dominant mediator of protective immunity induced by these recombinants. Antibody responses to vaccinia virus Ag and expressed influenza virus Ag were lower in titer in beta 2 mu(-/-) mice than in normal B6 mice, suggesting an influence of MHC class I complexes, CD8+ T cells, or their products on antibody production.

Animals↗