Rubella Virus Vaccine Strain Research Articles

Study of the teratogenicity of the vaccine strain of the Rubella virus «Orlov-V» (Matonaviridae: Rubivirus: Rubella virus) in experience on rhesus macaques.

Rubella virus has pronounced teratogenic properties that can cause generalized and persistent intrauterine infection of the fetus. As a result, the control of the loss of teratogenicity inherent in «wild-type» virus strains is a necessary stage of a preclinical study of the vaccine strain for a live attenuated rubella vaccine.The purpose of the study is to comprehensively study the teratogenic properties of the vaccine strain of rubella virus «Orlov-V» in the experiment on rhesus macaques. Seronegative to rubella virus female rhesus macaques in early pregnancy at the age of 4-7 years (n = 13) were used in the experiment. Animals of the experimental group (n = 9) received single immunization intramuscularly with a preparation from the «Orlov-V» strain. The control group of the monkeys (n = 3) were immunized with a commercial vaccine containing Wistar RA27/3 strain. The female of the control group (n = 1) was injected with a solvent used in the rubella vaccine. Study of possible teratogenic properties of vaccine strains of rubella virus was carried out using a complex of clinical, immunological, pathomorphological and virological methods. Clinical observations were made within 3 months after the monkeys' birth. Determination of antibody titers in the blood serum of immunized monkeys was performed in HI test on the 28th-30th day after infection. The ELISA method was applied to determine IgM antibodies in the blood serum of newborns within the first month of life. Detection of rubella virus RNA was performed by PCR with electrophoretic detection of amplicons. No markers of congenital rubella infection were found in infants born from monkeys vaccinated during the pregnancy. It is shown that PCR can be an informative method to confirm the absence of teratogenic properties of vaccine strains of rubella virus. The obtained data demonstrated that vaccine strains of the «Orlov-V» rubella virus and Wistar RA27/3 have lost their teratogenic properties. The possibility of using an alternative strategy for preclinical assessment of specific safety of antiviral vaccines including a complex of clinical, immunological, pathologic and virological methods instead of the classical pathologic method is discussed. The results obtained in this study showed the absence of teratogenic properties and high immunogenic activity of the vaccine strain of rubella virus «Orlov-V».

Rubella virus-associated chronic inflammation in primary immunodeficiency diseases.

The aim of this article is to summarize recent data on rubella virus (RuV) vaccine in chronic inflammation focusing on granulomas in individuals with primary immunodeficiencies (PIDs). The live attenuated RuV vaccine has been recently associated with cutaneous and visceral granulomas in children with various PIDs. RuV vaccine strain can persist for decades subclinically in currently unknown body site(s) before emerging in granulomas. Histologically, RuV is predominately localized in M2 macrophages in the granuloma centers. Multiple mutations accumulate during persistence resulting in emergence of immunodeficiency-related vaccine-derived rubella viruses (iVDRVs) with altered immunological, replication, and persistence properties. Viral RNA was detected in granuloma biopsies and nasopharyngeal secretions and infectious virus were isolated from the granuloma lesions. The risk of iVDRV transmissibility to contacts needs to be evaluated. Several broad-spectrum antiviral drugs have been tested recently but did not provide significant clinical improvement. Hematopoietic stem cell transplantation remains the only reliable option for curing chronic RuV-associated granulomas in PIDs. Persistence of vaccine-derived RuVs appears to be a crucial factor in a significant proportion of granulomatous disease in PIDs. RuV testing of granulomas in PID individuals might help with case management.

Live rubella virus vaccine long-term persistence as an antigenic trigger of cutaneous granulomas in patients with primary immunodeficiency

Granulomas may develop as a response to a local antigenic trigger, leading to the activation of macrophages and T-lymphocytes. Primary immunodeficiency (PID) is associated with the development of extensive cutaneous granulomas, whose aetiology remains unknown. We performed high-throughput sequencing of the transcriptome of cutaneous granuloma lesions on two consecutive index cases, and RT-PCR in a third consecutive patient. The RA27/3 vaccine strain of rubella virus—the core component of a universally used paediatric vaccine—was present in the cutaneous granuloma of these three consecutive PID patients. Controls included the healthy skin of two patients, non-granulomatous cutaneous lesions of patients with immunodeficiency, and skin biopsy samples of healthy individuals, and were negative. Expression of viral antigens was confirmed by immunofluorescence. Persistence of the rubella vaccine virus was also demonstrated in granuloma lesions sampled 4-5 years earlier. The persistence of the rubella virus vaccine strain in all three consecutive cutaneous granuloma patients with PID strongly suggests a causal relationship between rubella virus and granuloma in this setting.

Physiological parameters of Macaca fascicularis immunized with anti-rubella vaccine with germanium-based adjuvants.

Clinical status, hematological and biochemical parameters, and allergenic activity of organogermanium compounds used as adjuvants in complex with preparation from Orlov rubella virus vaccine strain and reference commercial anti-rubella vaccine based on Wistar RA 27/3 strain were studied on Macaca fascilcularis of both genders. Physiological parameters of monkeys immunized with the Russian and foreign rubella virus vaccine strains with and without adjuvants did not differ. The adjuvants were inessential for the safety of vaccines (absence of toxicity, reactogenic activity, or allergenic activity) in preclinical studies on lower primates.

Semliki Forest virus vectors expressing the H and HN genes of measles and mumps viruses reduce immunity induced by the envelope protein genes of rubella virus

A Semliki Forest virus (SFV) recombinant particle vaccine vector was constructed expressing the viral E1 and E2 envelope proteins of the RA27/3 vaccine strain of rubella virus. This vector induced high titres of antibody after intramuscular administration to Balb/C mice, both following initial vaccination and a boost 4 weeks later. This occurred for antibody as measured by ELISA and as measured by a latex agglutination test. However, co-administration of similar particles expressing the measles virus H protein and the mumps virus HN protein with the rubella protein expressing vector resulted in reduction of the anti-rubella immune response.

Mapping of genetic determinants of rubella virus associated with growth in joint tissue.

Rubella virus (RV) strains vary in their abilities to replicate and persist in cell cultures derived from human joint tissue (synovial cells [SC]), and this arthrotropism appears to be linked to their association with joint symptoms in vivo. In order to map the genetic determinants of arthrotropism, an infectious clone of the Cendehill vaccine strain of RV was constructed, as well as two chimeric clones containing cDNAs from both Cendehill and Therien (wild-type) strains. Replacement of the entire structural gene region of Therien in the infectious clone pROBO302 with the corresponding region of Cendehill did not affect growth in SC. A further observation that Cendehill bound equally well to SC and the permissive Vero cell line indicated that restriction was not at the level of receptor binding, a function of the envelope proteins. Mutations that affected growth in joint cells were mapped to two locations in the nonstructural gene region. The first of these (nucleotides 2803 and 6416) resulted in a 10-fold decrease in yield of progeny virus from SC. This region contained five mutations, at nucleotides 2829, 3060, 3164, and 3528 (near the carboxy terminus of P150 where the protease domain is located) and at nucleotide 4350 in p90. Further substitution of the sequence representing nucleotides 1 to 2803 to give a complete Cendehill infectious clone restricted growth in SC by a further 100-fold to less than 10 PFU/ml. This region contains three mutations, at nucleotides 34, 37, and 55, within the 5' stem-loop structure. In conclusion, the Cendehill-specific mutations believed to be determinants of joint cell growth are located in two regions, the 5' nontranslated region and in a sequence that encodes the carboxy-terminal region of p150 extending into the helicase domain of p90.

Genomic sequence of the RA27/3 vaccine strain of rubella virus.

The sequence of the genome of the RA27/3 vaccine strain of rubella virus (RUB) was determined. In the process, several discrepancies between the previously reported genomic sequences of two wild RUB strains (Therien and M33) were resolved. The genomes of all three strains contain 9762 nucleotides (nts), exclusive of the 3' poly A tract. In all three strains, the genome contains (5' to 3'), a 40 nt 5' untranslated region (UTR), an open reading frame (ORF) of 6348 nts that encodes nonstructural proteins, a 123 nt UTR between the two genomic ORFs, a 3189 nt ORF that encodes the structural proteins, and a 62 nt 3' UTR. The 5' end of the subgenomic RNA was found to correspond to a uridine residue at nt 6436 of the genomic RNA. At the nucleotide level, the sequence of the three strains varied by 1.0 to 2.8%, while at the amino acid level, the sequence varied by 1.1 to 2.4% over both ORFs. The RA27/3 sequence will be of use in identification of the determinants of its attenuation, in vaccine production control and in development of second generation RUB vaccines based on recombinant DNA technology.

Use of synthetic peptides to map regions of rubella virus capsid protein recognized by human T lymphocytes.

Synthetic peptides (SPs), 18-29 amino acids long, representing selected sequences of rubella virus (RV) capsid (C) protein were used in lymphocyte proliferation assays to identify antigenic regions recognized by T lymphocytes from healthy RV-reactive adults. Four SPs, C(1-29), C(90-114), C(108-134) and C(255-300), stimulated proliferation of peripheral blood mononuclear cells and RV-specific T-cell lines from the same donors. C(1-29V), an SP analogue containing an RA27/3 RV vaccine strain sequence, stimulated higher levels of proliferation in T cells obtained from RV-vaccinated subjects than did the comparable wild-type (M33 strain) RV sequence.

Nucleotide sequence of the 24S subgenomic messenger RNA of a vaccine strain (HPV77) of rubella virus: comparison with a wild-type strain (M33)

A full-length cDNA clone for the 24S subgenomic mRNA of the vaccine strain (HPV77) of rubella virus has been isolated from a cDNA library made from the RNAs of infected cells. Starting from the first Met start codon, the 24S mRNA codes for a precursor protein of 1063 amino acids (aa). This precursor encodes a capsid protein of 300 aa, and two envelope proteins. El (481 aa) and E2 (282 aa). Both the El and E2 proteins are preceded by a stretch of 21 hydrophobic aa, characteristic of a signal peptide, and each has three putative glycosylation sites in the polypeptide chains. Comparison between the structural proteins of the vaccine and the wild-type (wt; M33) strains of rubella virus, revealed that the E2 protein of the vaccine strain differs, in its apparent M r , by approx. 3 kDa, from the wt strain. The difference could be due to decreased glycosylation of the vaccine strain E2 protein, as revealed by [ 3H]mannose incorporation studies. Five single-aa changes in the structural proteins occurred during the attenuation process, one each in the capsid and the E1 protein and three in the E2 protein. The change of Thr-412 → Ile in the E2 protein results in the loss of a putative glycosylation site at Asn-410, which offers a plausible explanation for decreased glycosylation of the E2 protein from the vaccine strain of rubella virus.

Nucleotide sequence of capsid, E2 and E1 protein genes of Rubella virus vaccine strain RA27/3.

Nucleotide sequence of capsid, E2 and E1 protein genes of Rubella virus vaccine strain RA27/3.

Failure of the RA 27/3 strain of rubella virus to induce intrinsic interference.

Unlike other strains tested, the RA 27/3 vaccine strain of rubella virus, attenuated in human fibroblasts, failed to inhibit superinfection with Newcastle disease virus (i.e. induce intrinsic interference). Since proteins of the input virion are known to lead to intrinsic interference, these may differ in the RA 27/3 strain from those in other rubella strains.

Noncontagiousness of the Cendehill vaccine strain of rubella virus from mother to newborn infant

Noncontagiousness of the Cendehill vaccine strain of rubella virus from mother to newborn infant

An Immunologic Marker Technique for the Cendehill Vaccine Strain of Rubella Virus

Abstract Rabbits were immunized with serial, intramuscular doses of low tissue culture passage“wild” and high passage “attenuated” strains of rubella virus. Rubella hemagglutination-inhibiting antibody was detected in animals immunized with two serial doses of 3 log10 TCD50/ml of low passage strains. However, no antibody was detected in animals immunized with 3 log10 TCD50/ml of the Cendehill vaccine strain of rubella virus. A reisolate virus from a person immunized with Cendehill rubella vaccine also had markedly reduced immunogenic properties in the rabbit. Experiments indicated that the Cendehill vaccine virus had a markedly decreased ability to infect rabbits by the intranasal route. The technique may be a useful marker test for this strain of rubella vaccine. The RA/27/3 and the HPV 77 rubella vaccine strains also had a reduced immunogenicity for rabbits but the results were too variable to be used as a marker test for these two strains.

A Controlled Family Study of Live, Attenuated Rubella-Virus Vaccine

In a family study of live, attenuated rubella-virus vaccine (HPV80), 85 susceptible placebo recipients were exposed to siblings with evidence of infection with the vaccine strain of rubella virus after inoculation. Of these placebo recipients a rise in titer of hemagglutination-inhibiting antibody developed in one. The type of antibody response and her clinical course indicated that vaccine virus infection had occurred. Although this response might have resulted from her having received vaccine by mistake, the possibility of intrafamilial spread of vaccine-strain virus must be seriously considered.

Persistent rubella virus infection in hamster lung cells.

Summary Cell cultures were established from the lungs of hamsters which had been previously infected intranasally with the judith strain or the HPV 77 vaccine strain of rubella virus. The hamster lung cells were cultured in vitro and produced infective rubella virus for at least 12 subcultures. The persistently infected cells had a lag phase of 48 hr compared to a lag phase of 24 hr in uninfected cells. Morphologically, the persistently infected cells were indistinguishable from uninfected cells. Treatment of the persistently infected cells with 25 µg./ml. amantadine, 0.15 µg./ml. actinomycin D or 250 µg./ml. hydrocortisone had no detectable effect on rubella virus yields. Infective cell counts indicated that between 5 and 50% of hamster lung cells were releasing infective rubella virus. The persistently infected cells were resistant to challenge with vaccinia virus and, to a lesser degree, with herpes simplex virus.