Persistence of naturally acquired cell-mediated immunity to rubella virus during the first trimester of pregnancy

We studied the persistence of naturally acquired cell-mediated immunity to rubella during early pregnancy. We compared lymphocyte transformation responses to phytohemagglutinin and rubella virus in 35 naturally immune women aged 17-37 years, in the first trimester of pregnancy, with 49 naturally immune age-matched nonpregnant controls. A significant lower lymphocyte transformation response to phytohemagglutinin was observed during the first trimester of pregnancy (P = 0.008), but lymphocyte transformation responses to rubella virus were not significantly different (P = 0.901). These data indicate that, in naturally immune women, cell-mediated immunity to rubella virus is not significantly altered by the physiological changes in early pregnancy.     

Age related differences in cellular immune response to vaccine induced rubella infection.

Employing the techniques of in-vitro lymphocyte transformation (LTF) and using Putnam strain of rubella virus as the antigen, the development of rubella specific cellular immune response was studied in different age groups of rubella seronegative normal subjects at various intervals after subcutaneous administration of HPV=77/DE5 live attenuated rubella vaccine. The rubella specific lymphocyte response in children ranging in age from two to twelve years was characterized by significant LTF activity at two months, followed by a gradual decrease. The response in adult subjects 18 to 35 years of age showed a slight delay initially in the appearance and the maximum LTF activity appeared to be 3--4 fold lower (P less than 0.01) than observed in the children. No difference was observed in the maximum antibody titers to rubella virus between these two groups of subjects. These observations suggest that the age related differences in the lymphoproliferative responses might be associated with adverse effects which are known to occur more frequently in adolescent and older patients than in childhood population after vaccine induced rubella infection.     

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

Cryo-electron tomography of rubella virus

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses.     

风疹病毒松叶株衣壳蛋白C基因稳定性研究

目的研究风疹病毒(Rubella virus,RV)疫苗松叶株(Matsuba)衣壳蛋白(Capsid Protein)的基因稳定性及遗传特征,探讨其功能结构及生物学活性在病毒传代过程中的变化特点。方法利用RT-PCR方法扩增风疹病毒Matsuba株14、16、17、18、23代次C基因序列,测序后进行序列比对分析,并将各代次病毒的C基因与风疹病毒疫苗株Matsuba(GenBank登陆号:AB588193)及其他风疹病毒株C基因序列进行同源性分析。结果风疹病毒Mat-suba株传代病毒C基因在传代过程中核苷酸及氨基酸均未发生变异;各代次病毒与AB588193核苷酸及氨基酸序列完全一致,各关键功能区未发生变异;Matsuba株与18株风疹病毒核苷酸相似性在90.2%～99.9%之间。结论风疹病毒Matsuba疫苗株C基因遗传特性非常稳定,与生物学作用相关的区域未发生传代改变。从分子水平证明Matsuba疫苗株毒种及其生产的疫苗具有安全性。     

2001～2011年上海市风疹病毒分子流行病学研究

本研究对2001～2011年本实验室保存的血清标本检测结果为麻疹IgM阴性,风疹IgM阳性病例相对应的咽拭子标本进行风疹病毒（Rubella virus,RV）分离,用RT-PCR方法对细胞培养产物鉴定后扩增病毒E1基因,扩增产物用于核苷酸序列测定,并进行分子流行病学分析。结果表明,60份咽拭子标本共分离到31株RV,获得27株RV的739nt（nt8 731～nt9 469）核苷酸序列,系统同源性分析表明,27株RV分离株分别属于两个不同的基因型,除分离自2011年的11009株、11052株和11106株为2B基因型外,其他分离株均为1E基因型。27株RV分离株大部分的核苷酸突变为无义突变,氨基酸序列高度保守。24株1E基因型分离株中大部分毒株氨基酸序列完全一致,自2001年以来可能有来自同一传播链的RV在持续传播。本研究首次监测到2B基因型RV2011年开始在上海流行,经GenBank核苷酸序列比对发现,其与越南、日本、阿根廷等国家近几年的RV分离株核苷酸序列同源性达99%。由于以前对风疹的监测较少,尚不能证明其来源。     

The development and standardization of an antigen detection ELISA for rubella virus grown in rabbit cells

A double antibody sandwich ELISA for the detection of rubella virus antigen was developed and standardized. Commercially available antisera were chosen in order to make the assay readily available. Antigen detection gave an excellent correlation to titers obtained by examination of cytopathogenic effect (CPE, r = 0.986). Replication of rubella virus grown in rabbit cells was identified with CPE and positive ELISA appearing within a difference of +/- one day. ELISA provided an objective detection of rubella virus which is often difficult by the reading of CPE. The method was found to be both sensitive and reproducible and facilitated work in rubella virus control involving a large number of virus titrations.     

Effect of selective vaccination on rubella susceptibility and infection in pregnancy.

The effect of school and adult vaccination on susceptibility to rubella in women of childbearing age was assessed in the Manchester area, where the population attending antenatal clinics is over 40 000 a year. Between 1979 and 1984 the proportion susceptible fell from 6.4% to 2.7%. In 1984, 4.2% of nulliparous women were susceptible compared with 1.4% of women in their second or subsequent pregnancy. Eighty five per cent of pregnant women screened and found to be non-immune were vaccinated post partum before leaving hospital. Requests for prevaccination screening of non-pregnant women increased in response to a national campaign and at the time of local outbreaks of rubella but only two thirds of those found to be nonimmune were subsequently vaccinated. During 1983 and 1984 infection was confirmed in 57 pregnant women--2% of those non-immune. Selective vaccination has reduced susceptibility to rubella in the childbearing population, but it is suggested that mass vaccination of children of both sexes should be added to the existing policy to control circulation of wild rubella virus and reduce the risk of infection to pregnant women who remain susceptible.     

Absence of cell-mediated immunity to rubella virus 5 years after rubella vaccination.

The long-term effectiveness of rubella vaccination in childhood is particularly important because the ultimate goal of immunization is the prevention of infection during pregnancy. Of 25 healthy children tested 4 to 5 years after rubella vaccination, 19 showed no evidence of cell-mediated immunity (CM) to rubella virus despite the presence of hemagglutination-inhibition or complement-fixation antibodies or both. Twenty-two of 25 seropositive, naturally infected young adults showed evidence of CMI. These results indicate that fetuses of women who have been vaccinated against rubella may not be protected against damage by wild rubella infection during the pregnancy, when CMI is physiologically depressed.     

Concentration and purification of rubella virus using monolithic chromatographic support

The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved.     

Retrospective diagnosis of congenital rubella.

One hundred and five children and adolescents with impaired hearing and 19 with impaired vision underwent in vitro tests (lymphocyte responsiveness and serum antibody to rubella) for retrospective diagnosis of intrauterine rubella. Tests yielded results consistent with intrauterine rubella in 30 (29%) of the patients with impaired hearing but only one (5%) of those with impaired vision. In addition, the reported incidence (10.8%) of rubella as a cause of deafness was obtained by questioning parents before the tests. Of 27 patients with impaired hearing of unknown aetiology but reported rubella contact during the pregnancy, seven (26%) had test results consistent with intrauterine rubella. The incidence of intrauterine rubella as a cause of deafness is probably underestimated when the diagnosis is based on the presence of several classic features.     

Cytokine profile after rubella vaccine inoculation: evidence of the immunosuppressive effect of vaccination

BACKGROUND AND AIM: Immunization with live virus vaccines may cause an immunosuppression with lymphopaenia, impaired cytokine production and defective lymphocyte response to mitogenes. These abnormalities were described in subjects vaccinated against measles. This study was performed to analyse the host immune response related to immunosuppression in subjects vaccinated with live attenuated rubella vaccine. METHODS: Eighteen schoolgirls, aged 11-13 years, were vaccinated with live attenuated rubella vaccine Rudivax. Before immunization, and 7 and 30 days after, peripheral blood was collected. Cellular fractions were subjected to flow cytometric analysis, and the lymphocyte response to phytohaemagglutinin was investigated. Plasma samples were analysed for cytokines (interleukin (IL)-4, IL-10, tumour necrosis factor-alpha, and interferon-gamma) by enzyme-linked immunosorbent assay techniques. RESULTS: On day 7 after vaccination, the number of each lymphocyte subset was decreased; however, only for CD3 and CD4 lymphocytes has a significant reduction been shown. On the contrary, tumour necrosis factor-alpha and IL-10 levels markedly increased and amounted to its maximum on day 30. Simultaneously, a significant reduction in plasma interferon-gamma and a profound decrease of the lymphocyte response to phytohaemagglutinin were shown. The changes were accompanied with marked elevation of plasma IL-4. CONCLUSIONS: Our data indicate that the vaccination with live attenuated rubella vaccine results in moderate but sustained immune disturbance. The signs of immunosuppression, including defective lymphocyte response to mitogene and impaired cytokine production, may persist for at least 1 month after vaccination.     

Prevention of congenital rubella.

The vaccine of choice for rubella vaccination is considered to be RA 27/3, based on frequency of side effects, duration of immunity, antigenic potential and rate of reinfection by wild virus. The most appropriate individuals to be vaccinated are prepubertal schoolgirls and susceptible members of other high-risk groups, and a nationwide immunization program is suggested. Premarital determination of rubella-immune status should be mandatory for all women of childbearing age. A favourable cost/benefit ratio for rubella vaccination seems highly probable. The use of a rubella "fact sheet" to provide education and information for those at risk is strongly recommended.     

Guide to the management of rubella problems.

Despite the availability of rubella vaccine the incidence of the congenital rubella syndrome has been increasing in certain regions of Canada. Perhaps this is not surprising in view of the known irregular cyclic activity of rubella virus in a community and the fact that the percentage of seropositive women of childbearing age has not changed appreciable since the vaccine was introduced. Clearly vaccine is not being administered to sufficient numbers of women at risk. Until a much higher percentage of women of childbearing age possess rubella antibody, the costly problem of congenital rubella syndrome is likely to be with us. Common rubella problems relate to four categories: the exposed pregnant woman, laboratory diagnosis, the infant with suspected congenital rubella and the vaccine. One of the most common questions about the vaccine is the following: Can recently vaccinated individuals disseminate vaccine and infect seronegative contact? The answer is No.     

Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay.

The quantitative PCR infectivity assay is a combination of virus propagation and quantitative PCR. Previously [Schalk JAC, van den Elzen C, Ovelgonne H, Baas C, Jongen PMJM. Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR. J Virol Methods 2004;117:179-87.], we used this assay to estimate the titer of infectious measles virus in trivalent, live, measles, mumps, rubella vaccines (MMR). Here we describe the further improvement and development of the assay for simultaneous potency estimation of measles, mumps and rubella viruses. The potency of measles and mumps virus is estimated within one assay after 1 day of cell culture. The potency of rubella virus is estimated in a separate assay after 2 days of cell culture. Compared to conventional CCID50 and plaque assays, the quantitative PCR infectivity assay has the advantage in being fast because the assay is not dependent on the formation of cytopathic effect. Furthermore assay design is simplified: serological neutralization can be omitted because PCR is virus-specific and, under the conditions used, the individual components of trivalent measles, mumps, rubella vaccines do not interfere with each other. The assay was validated and compared to the performance of a plaque assay.     

Reinfection after rubella and congenital polymalformation syndrome

A case is reported of a term newborn with intra uterine growth retardation and numerous malformations such as complex heart disease, abnormalities of distal limbs, cleft palate. Death occurred after two days. The diagnosis of rubella embryopathy was confirmed by the following criteria: a high level of rubella antibodies in mother and newborn (1/1000) an isolation of rubella virus from the infant's urine. Diagnosis of rubella after reinfection was documented by a high level of antibodies in the mother three years before this pregnancy. Other observations reported in literature confirm the extreme rarity of congenital rubella after reinfection.     

Can we prevent an increase in the incidence of congenital rubella syndrome in the next decade?

The immunity to rubella of 115 girls aged 10 to 14 years was tested in 1978. The proportion of girls found to be immune was 80%, similar to rates in the prevaccination era. Nearly half of the immunity was from documented vaccination, and the other half was presumably from infection with wild rubella virus. The vaccination failure rate was 12%. Because of declining immunity to rubella of women of child-bearing age, detecting low levels of immunity in these women is becoming increasingly important. Immunization of 12- to 15-month-old children has not been effective. Vaccinating all girls 10 to 12 years old would likely be the most effective method of preventing an increase in the incidence of congenital rubella syndrome in the next decade.     

Congenital rubella affecting an infant whose mother had rubella antibodies before conception.

A woman who had had high titres of rubella antibodies some months before she became pregnant gave birth to an infant in whom congenital rubella was confirmed at 4 months. Rubella haemagglutination inhibition tests, complement fixation tests, and immunofluorescence tests with anti-human IgG were carried out on sera from the mother. Rubella antibody titres in sera obtained in March 1971, seven and a half months before conception, were equivalent to 400 units, which is usually taken as indicating good immunity. Rubella virus was isolated from the baby''s nose and throat in July 1973 but was not isolated from a cervical swab taken from the mother in December 1973; tests of her immunological competence did not show any definite abnormality. The presence of high levels of rubella haemagglutination inhibition antibodies does not invariably confer immunity or exclude the possibility of congenital rubella in a subsequent pregnancy.     

Characteristics of serially propagated monkey kidney cell cultures with persistent rubella infection

Maassab, H. F. (University of Michigan, Ann Arbor), and J. A. Veronelli. Characteristics of serially propagated monkey kidney cell cultures with persistent rubella infection. J. Bacteriol. 91:436-441. 1966.-A persistent infection of LLC-MK(2) cells with rubella virus has been established and maintained for over 3 years. This "carrier culture," designated as LLC-MK(2)-RAL, possesses distinct morphological and biological characteristics when compared with the original uninfected LLC-MK(2) line. The mechanism of viral persistance has not been entirely elucidated, but available data suggest a regulated infection with transmission of the virus directly from cell to cell or through cell division. Interferon was isolated from RAL (rubella-associated line) culture, which explains partly the wide spectrum of resistance to superinfecting viruses. Amantadine, although inhibiting cultures of LLC-MK(2) cells infected with rubella virus, failed to cure the "carrier culture."     

Influence of albumin on rubella virus hemagglutination and the hemagglutination-inhibition test.

The HEPES-saline-albumin-gelatin (HSAG) diluent found optimal for agglutination of fowl erythrocytes by rubella virus antigen is also optimal for agglutination of trypsin-treated human group O cells. Albumins from different commercial sources, however, can have varying inhibitory effects on rubella hemagglutination titers. This can have a significant effect on the hemagglutination-inhibition test since antibody titers measured by this procedure are related to the amount of antigen used.