人3型副流感病毒疫苗的研究进展

人3型副流感病毒是一种主要感染人类肺部上皮细胞的副黏病毒,可引起肺炎和支气管炎,在婴幼儿和免疫力低下的成人中有较高的发病率。经过多年的研究,对人3型副流感病毒疫苗的研究取得了重要的进展,但还没有有效的抗病毒药物和批准的疫苗上市。目前研究主要集中在减毒活疫苗及亚单位疫苗等,对人3型副流感病毒当前疫苗的研究情况做简要的综述。     

Characterization of bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (PIV-3) has a buoyant density of 1.197. The RNA of PIV-3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self-anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 X 10(6), being slightly smaller than Sendai virus RNA (47S, 5.3 X 10(6)). PIV-3 has 5 main structural proteins, of which 2 are glycoproteins. The molecular weights of protein 1, protein 2, protein 3, glycoprotein 1, and glycoprotein 2 were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000, respectively. Protein 2 was suggested to be nucleocapsid protein.     

人3型副流感病毒减毒活疫苗的研究进展

人副流感病毒(human parainfluenza viruses,HPIVs)是引起婴幼儿支气管炎和肺炎的主要病原体,人3型副流感病毒(human parainfluenza viruses type 3,HPIV-3)是HPIVs中最主要的型别。目前尚无针对HPIVs的上市疫苗和有效的抗病毒药物,研发减毒活疫苗是目前开发HPIVs疫苗的方向。本文主要对HPIV-3减毒活疫苗的研究进展作一综述。     

人3型副流感病毒及其疫苗的研究进展

人3型副流感病毒(Human Parainfluenza Virus type 3,HPIV-3)是引起婴幼儿严重细支气管炎及肺炎等下呼吸道疾病的主要病原体,其在发达国家和发展中国家都造成了沉重的疾病负担。迄今,对HPIV-3感染的预防和治疗都还没有有效的疫苗和药物,因此WHO将HPIV-3疫苗列为未来重点研发的疫苗。近年来,随着重组技术和反向遗传学的发展,HPIV-3疫苗的研制取得了重要进展,部分疫苗已进入临床评价阶段。就HPIV-3的生物学特性如病毒结构特征、复制过程、流行病学特征,以及近年来传统冷适应减毒活疫苗、亚单位疫苗、以反向遗传学为基础的新型减毒活疫苗的研制成果及临床试验进展作简要综述。     

Expression of the surface glycoproteins of human parainfluenza virus type 3 by bovine parainfluenza virus type 3, a novel attenuated virus vaccine vector

Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39 degrees C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37 degrees C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses.     

Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein.

Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) glycoprotein of human type 3 parainfluenza virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which participate in neutralization. Competitive binding assays indicated that the 14 neutralization epitopes are organized into three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB) and that the 6 nonneutralization epitopes form four sites (D, E, F, and G). Most of the neutralizing MAbs were involved in nonreciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralization epitopes. Fusion-inhibition and complemented-enhanced neutralization assays indicated that antigenic sites AB, B, and C may correspond to functional domains of the F molecule. Our results indicated that antibody binding alone is not sufficient for virus neutralization and that many anti-F MAbs neutralize by mechanisms not involving fusion-inhibition. The degree of antigenic variation in the F epitopes of clinical strains was examined by binding and neutralization tests. It appears that PIV3 frequently develops mutations that produce F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies.     

Lentiviral vectors pseudotyped with envelope glycoproteins derived from human parainfluenza virus type 3

We describe the generation of lentiviruses pseudotyped with human parainfluenza type 3 envelope (HPIV3) glycoproteins. Lentivirus particles, expressed in 293T/17 cells, incorporate HPIV3 hemagglutinin-neuraminidase (HN) and fusion (F) proteins into their lipid bilayers and are able to transduce human kidney epithelial cells and polarized MDCK cells. Neuraminidase, AZT, and anti-HPIV3 antisera block transduction, which is consistent with lentiviral-mediated transduction via sialated receptors for HPIV3. Our findings show that HPIV3 pseudotyped lentiviruses can be formed and may have a number of useful properties for human gene transfer.     

Mechanism of interference mediated by human parainfluenza virus type 3 infection

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.     

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.     

Specific inhibition of granzyme B by parainfluenza virus type 3.

T-cell-mediated cytotoxicity is an important means of defense against viral pathogens; however, several viruses possess mechanisms to disrupt cytotoxicity, thereby allowing them to avoid immune clearance. These viruses have been shown to inhibit cytotoxicity by interfering with the capacity of T lymphocytes to specifically recognize infected cells. An alternative mechanism for virally induced cytotoxic dysfunction is identified in this report. We show that parainfluenza virus type 3, a negative-stranded RNA virus, can inhibit cytotoxicity by causing a defect in the cytotoxic effector apparatus. This defect is identified as a selective inhibition of granzyme B mRNA.     

Intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides.

The intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides was examined by polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts under reducing conditions. All of the virion structural proteins were detected in cell extracts, including: L, 180,000 molecular weight (180K); P, 83K; HN, 69K; NP, 66K; F0, 60K; F1, 51K; and M, 38K. P and NP were phosphorylated. HN and F were glycosylated. The kinetics of intracellular viral protein synthesis did not detect any early or late proteins. Pulse-chase experiments failed to detect any precursor-product relationships. No nonstructural proteins were detected.     

Natural killer cells regulate T-cell proliferation during human parainfluenza virus type 3 infection

Human parainfluenza virus type 3 (HPIV3) is a major respiratory pathogen in humans. Failure to induce immunological memory associated with HPIV3 infection has been attributed to inhibition of lymphocyte proliferation. We demonstrate that the inability of mixed lymphocytes to respond to virally infected antigen-presenting cells is due to an interleukin-2-dependent, nonapoptotic mechanism involving natural killer (NK) cells and their influence is exerted in a contact-dependent manner. These results suggest a novel regulatory mechanism for NK cells during HPIV3 infection, offering an explanation for viral persistence and poor memory responses.