Rubella Vaccines


RUBELLA

“German measles is extremely infectious. People catch it very easily. And there’s one thing about it that you’ve got to remember. If a woman contracts it in the first four months of pregnancy it may have a terribly serious effect. It may cause an unborn child to be born blind or to be born mentally affected.” Agatha Christie, in “The Mirror Crack’d From Side To Side”

First described in the late 18th century as a mild exanthematous disease of children and young adults, rubella became prominent in 1941 as a cause of congenital defects in the fetus after maternal infection during pregnancy. The development and use of a live attenuated vaccine has controlled rubella in developed countries during the last 50 years, even enabling elimination in many European countries and the entire Western Hemisphere. More recently, use of the vaccine has been extended to Asia and Africa. Nevertheless, congenital rubella continues to be an important problem in countries not using vaccine.

The first researchers to distinguish the disease from other exanthems were German physicians; hence, the common English language eponym German measles . In 1814, a British physician named George Maton described an outbreak of a rash disease. In describing an outbreak in a boys’ school in India in 1866 Henry Veale coined the term rubella , a Latin diminutive meaning “little red.” For the next 100 years, rubella received scant attention, but in 1941, Norman McAlister Gregg, an Australian ophthalmologist, linked congenital cataracts to maternal rubella. Gregg had noticed an unusual number of infants with cataracts coming to his practice, and he was curious enough to investigate. It is said that a crucial clue was a conversation he overheard in his waiting room between two mothers who were discussing the rubella they both had sustained in pregnancy during the Australian outbreak of 1940. After several years of inattention and skepticism, Gregg’s original observation was followed by reports of Australian, Swedish, American, and British epidemiologists and teratologists confirming the role of rubella in congenital cataracts and also noting the simultaneous association of heart disease and deafness in the infants. Thus, the characteristic congenital rubella triad was established.

During the next 20 years, efforts were made to isolate the causative agent and obtain statistics on the risk of fetal abnormality after maternal rubella. The disparity in estimates stemmed from the absence of a definitive diagnostic test and consequent misdiagnosis of rubella in the mother. In late 1962, a breakthrough came in the form of the first isolations of rubella virus by Weller and Neva in Boston and by Parkman, Beuscher, and Artenstein in Washington, DC. Weller and Neva detected the presence of rubella virus by cytopathic effect in human amnion cells, whereas Parkman, Beuscher, and Artenstein developed a technique dependent on interference with the growth of enteroviruses in African green monkey kidney (AGMK) cell culture.

Meanwhile, a pandemic of rubella started in Europe in the 1962–1963 season, with spread to the United States in 1964–1965. As a result, from 1964 to 1966, thousands of pregnancies were affected by rubella, leaving behind a wake of medically induced abortions and abnormal infants. , The pandemic led to the recognition of an expanded congenital rubella syndrome (CRS), which added hepatitis, splenomegaly, thrombocytopenia, encephalitis, mental retardation, and numerous other anomalies to the already described deafness, cataracts, and heart disease. , , The pandemic also made it obvious that a vaccine was needed, and many groups set to work. Between 1965 and 1967, several attenuated rubella strains were developed and reached clinical trials. , , In 1969 and 1970, rubella vaccine entered commercial use in Europe and North America. Since the late 1970s, vaccination has had a major impact on the epidemiology of rubella and CRS.

BACKGROUND

Clinical Description

Acquired Rubella

Rubella is spread by large particle aerosols; primary implantation and replication occurs in the nasopharynx. The incubation period of rubella is 14–23 days, with most patients developing a rash 14–17 days after exposure. During the first week after exposure, there are no symptoms, but in the second week, lymphadenopathy may be noted, particularly occipital and postauricular, and virus cultures reveal rubella virus in the nasopharynx. Later in the second week, virus appears in the blood. About this time, there may be a prodromal illness consisting of low-grade fever (<39.0°C), malaise, and mild conjunctivitis. If not already present, lymphadenopathy is likely to develop.

At the end of the incubation period, a maculopapular erythematous rash appears on the face and neck in about two thirds of cases, but the clinical to subclinical ratio is variable. The rash may be difficult to detect, particularly on pigmented skin, and is more prominent after hot showers or baths. During a course of 1–3 days, the rash spreads downward and begins to fade. Pharyngeal virus excretion is present in high titer up to 4 days after rash onset, and virus excretion in the pharynx and urine may continue for another 1–2 weeks, but viremia ends with the appearance of antibodies, which is more or less simultaneous with the onset of the rash. Fig. 54.1 illustrates the evolution of acquired rubella.

Fig. 54.1, Sequence of events in acquired rubella infections, showing the relationship between onset of rash and other clinical symptoms and recovery of rubella virus from diagnostic specimens. Serum is noted by the dotted line; pharynx is noted by the solid line.

Although acquired rubella is thought of as a benign disease, arthralgia and arthritis commonly are observed in adults, particularly women, associated with virus replication or latency in the synovia. , Chronic arthritis has been reported after rubella infection, although the evidence is weak. , Other less-common complications are thrombocytopenia and encephalitis, which may be fatal. , Encephalitis has been reported to occur in approximately one in 6,000 cases and is of the postinfectious type, although the limited available pathologic data show little evidence of demyelination. However, the incidence of encephalitis was one in 1,600 cases of rubella in a Japanese outbreak ; and in the South Pacific islands of Tonga and Samoa, the incidence was estimated as between one in 500 and one in 1,000 cases. In 2011–12 in Tunisia, 280 cases of rubella were confirmed, including 39 cases of encephalitis that occurred mainly in adolescents and adults. Another report from the 2011–2012 rubella outbreak in Tunisia described nine cases of encephalitis, most of which were in children. A study done in China between 2010 and 2012 using a multiplex reverse transcription–polymerase chain reaction (PCR) showed that 3.3% of viral encephalitis was caused by rubella. In addition, there is a rare late syndrome of progressive rubella panencephalitis that is inexorable and may also be seen in congenital infection. , By analogy to subacute sclerosing panencephalitis caused by measles, progressive rubella panencephalitis is thought to be related to persistence of viral antigens. Guillain-Barré syndrome after rubella has also been reported, although the causal relationship is uncertain. Antibodies to rubella and measles structural proteins are elevated in autoimmune chronic active hepatitis, but there is no evidence for the presence of rubella virus in this disorder.

Rubella virus is one of the agents that causes Fuchs heterochromatic anterior uveitis. This condition is a chronic inflammatory syndrome that may lead to cataracts and glaucoma. Rubella virus RNA and high-titer antibodies have been demonstrated in intraocular fluid of patients with this condition. Intraocular rubella virus was detected in a 28-year-old survivor of congenital rubella syndrome, indicating the ability of the virus to persist and cause Fuchs uveitis. The prevalence of the syndrome has decreased in the United States since the introduction of vaccination.

Congenital Rubella Syndrome

Rubella is the archetypical fetal infectious pathogen (see “Pathogenesis as It Relates to Prevention”). Because all organs of the fetus are affected, it is not surprising that CRS comprises a lengthy list of abnormalities, including anatomic changes resulting from interference with organogenesis and inflammatory effects involving organs such as the liver and spleen ( Table 54.1 ). Histopathological study of several congenital rubella syndrome cases showed widespread presence of rubella antigen in fibroblasts lining the heart and blood vessels, alveolar macrophages, progenitor cells of the outer granular layer of the brain, as well as capillary endothelium in the placenta. Rubella virus replicates in the nasopharynx of affected infants and presents a risk to others. In a Japanese study, 17% of infected infants were still excreting rubella virus 1 year after birth.

TABLE 54.1
Prominent Clinical Findings in Congenital Rubella Syndrome
Cataracts
Retinitis
Microphthalmia
Glaucoma
Cochlear deafness
Central auditory imperception
Patent ductus arteriosus
Peripheral pulmonic artery stenosis
Encephalitis
Microcephaly
Mental disability
Autism
Intrauterine growth retardation
Metaphyseal rarefactions
Hepatosplenomegaly
Thrombocytopenic purpura
Interstitial pneumonitis
Late:
Diabetes
Hypothyroidism
Modified from Cooper LZ, Preblud SR, Alford CA. Rubella. In: Remington JS, Klein JO, eds. Infectious Diseases of the Fetus and Newborn Infant . 4 th ed. Philadelphia, PA: WB Saunders; 1995:288.

The time of infection during gestation is important in relation to the fetal outcome. Infection in the first 2 months often leads to spontaneous abortion; if the pregnancy survives, serious ocular or cardiac disease is likely to result. In contrast, infection late in the first half of pregnancy is more likely to result in isolated hearing impairment. However, the relationship between gestational age and abnormality can be overemphasized because fetal infection, once established, spreads to all organs, and damage may be progressive. Rubella virus probably reaches the placenta through infected monocytes, where it replicates in many different types of cells, including cytotrophoblasts, endothelial cells, amniotic epithelium and cells in the decidua.

Table 54.2 demonstrates that organ specificity is only generally related to the stage of gestational infection with rubella virus. The most common congenital defects are sensorineural deafness, cataracts, pigmentary retinopathy, and patent ductus arteriosus; however, malformations of the pulmonary artery or its branches can be demonstrated in 78% of CRS patients in whom cardiac studies are done, and patent ductus arteriosus is found in 62%. In addition, myriad other abnormalities occur, including glaucoma, endocrinopathies such as diabetes, hyperimmunoglobulinemia M, microcephaly, and mental disability. Ocular defects are particularly varied, including abnormalities of the cornea, the lens, the retina, and uveitis. All infants with congenital cataracts should be screened for CRS, which will turn up many cases. An analysis of prospective studies for distribution of clinical manifestations of CRS is summarized in Table 54.3 .

TABLE 54.2
Age of Gestation at Time of Rubella in Relation to Abnormalities Observed
Month of Gestation Measured From Last Menstrual Period
0 a 1 2 3 4
Birth weight <2,500 g 0/1 b 9/21 9/21 10/18 0/2
<38 weeks gestation 0/1 5/21 2/21 4/18 0/2
Growth retardation 0/1 7/21 5/20 7/17 0/2
Ocular defects 0/1 14/21 9/21 9/18 0/2
Cardiac defects 0/1 17/21 13/21 6/18 0/2
Deafness 0/1 8/18 10/18 11/17 2/2
Mental retardation 1/1 7/20 7/20 8/16 0/2
Microcephaly 1/1 3/18 2/19 4/17 0/2

a Before conception.

b Ratio of number of patients with condition to total number for whom information is available. From Plotkin SA, Cochran W, Lindquist J, et al. Congenital rubella syndrome in late infancy . JAMA . 1967;200:435–441. Copyright 1967, American Medical Association .

TABLE 54.3
Frequencies of Selected Defects in Infants With Congenital Rubella Syndrome: Comparison of Data From Prospective Studies and Data Presented in a Standard Pediatric Infectious Disease Textbook
Clinical Manifestation No. of Studies Study Subjects (%) a Previously Reported Studies (%) b
Hearing impairment 10 68/113 (60) 80–90
Heart defect 9 45/100 (45)
Patent ductus arteriosus 3 9/45 (20) 30
Peripheral pulmonic stenosis 3 6/49 (12) 25 c
Microcephaly 3 13/49 (27) Rare
Cataracts 3 16/65 (25) 35
Low birth weight (<2,500 g) 2 5/22 (23) 50–85
Hepatosplenomegaly 6 13/67 (19) 10–20
Purpura 5 11/65 (17) 5–10
Mental retardation 2 2/15 (13) 10–20
Meningoencephalitis 3 5/49 (10) 10–20
Radiolucent bone 3 3/43 (7) 10–20
Retinopathy 3 2/44 (5) 35 c

a Number of infants with congenital rubella syndrome/number of study subjects (percentage in parentheses).

b Frequencies presented in textbook data.

c Includes pulmonary arterial hypoplasia, supravalvular stenosis, valvular stenosis, and peripheral branch stenosis. From Reef SE, Plotkin S, Cordero JF, et al. Preparing for elimination of congenital rubella syndrome (CRS): summary of a workshop on CRS elimination in the United States. Clin Infect Dis . 2000;31:85–95.

A comprehensive review of the retrospective and prospective studies estimated that hearing, cardiac, ocular, and intellectual manifestations were found, respectively, in 70%, 31%, 16%, and 6% of CRS infants. However, if mixed-cohort and infant-case series are included, the percentages of cardiac, ocular, and intellectual manifestations increase to 46%, 35%, and 40%, respectively. Various other estimates have been made of the incidence of fetal abnormalities after maternal infection. , Many prospective studies of the incidence of fetal rubella have accepted clinical diagnosis of rubella in the mother for case inclusion, but if only virologically confirmed maternal rubella is considered, the rate of transmission to the fetus during the first 10 weeks of pregnancy is as high as 90%. , Table 54.4 tabulates data from the two most reliable studies, one from United States and the other from United Kingdom. The first 12 weeks of pregnancy are clearly the most dangerous time for rubella infection in the mother. The incidence of fetal disease declines during the next 4 weeks, and in the 16th to the 20th weeks, only deafness has been reported as a complication. Preconceptional rubella rarely results in fetal infection, but rashes that occur within 12 days of the last menstrual period carry proven risk. , In an Irish epidemic, 71% of fetuses infected early in pregnancy were affected.

TABLE 54.4
Fetal Abnormality Induced by Confirmed Rubella at Various Stages of Pregnancy
Stage of Pregnancy (Week) United Kingdom Study (% Defective) a United States Study (% Defective) b
≤4 70
5–8 40
≤10 90
11–12 33
9–12 25
13–14 11
15–16 24
13–16 40
≥17 0 8 a

a Data from Miller E, Cradock-Watson JE, Pollock TM. Consequences of confirmed maternal rubella at successive stages of pregnancy. Lancet . 1982;2:781–784 .

b Data from South MA, Sever JL. Teratogen update: the congenital rubella syndrome. Teratology . 1985;31:297–307 .

Damage caused by congenital rubella infection is not always evident at birth: glaucoma may become apparent and late cataracts are possible. Retinal detachments and esophageal problems are common later in life. Of interest, autism is a feature of some late-onset neurologic disease in CRS patients. In addition, a variety of syndromes thought to be autoimmune are seen, including diabetes mellitus and thyroiditis. , ,

Diabetes developed in 12% to 20% of American, European, and Australian CRS survivors. , However, a 40-year follow-up of Japanese CRS patients showed that only 1.1% were diabetics. Although autoimmunity is involved in ordinary diabetes mellitus, a thorough study of CRS patients showed no increase in autoantibodies to pancreatic proteins.

Virology

The agent of rubella is a cubical, medium-sized (50–85 nm), lipid-enveloped virus with an RNA genome belonging to the Matonaviridae family and the genus Rubivirus . Although the other togaviruses are arthropod-borne, there is no evidence for such transmission of rubella. Frey has reviewed the virology of rubella. Apart from the complex lipid envelope derived from the host cell, rubella virus is composed of three proteins: two embedded in the envelope in the form of spikes (E1 and E2) and one (C) composing the capsid. E1 is a glycoprotein with neutralizing and hemagglutinating epitopes, while the function of the E2 glycoprotein is unclear. The three proteins, which have molecular masses of 60,000 kilodalton (kDa) (E1), 42,000–47,000 kDa (E2), and 30,000 kDa (C), are derived from a polypeptide of 110 kDa that is translated from a 245-kDa messenger RNA.

The positive-sense RNA genome of rubella virus contains approximately 9,800 nucleotides and is infectious. , The replication strategy of rubella virus is similar to that of the alphaviruses in that both full-length and subgenomic RNAs are produced, and it is from the subgenomic RNA that viral structural proteins are translated. Three other proteins are produced by the virus in infected cells but are not incorporated into the virion. The structural capsid protein interferes with cellular mitochondrial function, inhibiting transport of proteins into the mitochondria and causing morphologic alterations of those organelles. A receptor for the virus has not been identified definitively, but myelin oligodendrocyte glycoprotein may be involved in spread to the placenta and fetal brain.

There is only one serotype of rubella virus, and analyses of sequence variation among occidental isolates show high conservation of amino acid structure (0% to 3.3% differences), with less conservation of isolates from Asia (up to 7%). , However, there are two clades and 13 genotypes. , For rubella isolates collected before 2000, isolates from North America, Europe, and Japan are closely related to each other and form clade I, whereas clade II comprises some strains from China, Korea, and India. However, in China, between 2001 and 2007, there was a shift of genotypes toward predominance of Clade I genotypes 1E and 1F. Similarly, in France, genotype 1E has become predominant. Isolation of virus from cataracts in Indian CRS cases revealed genotype 2B. Of rubella isolates collected in recent years, four genotypes (1E, 1G, 1J and 2B) had wide geographic distribution, whereas others occurred sporadically or were geographically restricted. , Other studies identified the circulation of multiple genotypes, including in Africa, with 1B, 1E, 1G, and 2B being prominent. , Genotype 1G seems to be particularly prevalent in Africa. Genotype 2B predominates in India. The genetic differences between clades do not appear to translate into antigenic differences, despite amino acid changes of 3% to 6% in viral proteins. Moreover, isolates from CRS cases are not genetically distinct from isolates from acquired rubella. No significant antigenic drift has occurred in recent years, and isolates from vaccinees showed little variation from vaccine strains. However, recent data show many intercontinental transfers of clades I and II. Because clade II was clearly of Asian origin, and clade I isolates from Asia are highly variable, an Asian origin of rubella virus has been suggested. Genetic analysis allowed the confirmation of the epidemiologic data that implicated spread of endemic rubella strains from Greece to the United Kingdom in 1999.

Two zoonotic viruses genetically related to rubella virus have been identified. One, called Rustrela, was found in various animals in a German zoo. Another, called Ruhugu, was isolated from bats in Uganda, and showed clear genetic relationship to rubella. How Ruhugu evolved to rubella is uncertain, but this finding suggests that, as for other human viruses, pathogens for humans evolved from viruses of animals. ,

Rubella virus grows in many different primary, semicontinuous, and continuous cells of mammalian origin. In human amnion cells, it produces a subtle cytopathic effect. More pronounced cytopathic effects, sufficient to allow plaque formation, are produced in continuous cell lines, such as rabbit kidney (RK13) and baby hamster kidney (BHK-21). Even in those cell lines, fresh isolates frequently are not cytopathogenic and require adaptation by serial passage. High passage generates defective interfering RNA and particles. Fig. 54.2 describes the replication of rubella virus in cell culture.

Fig. 54.2, Model of the RUBV life cycle. At the top: E2 (blue), E1 (red), and nucleocapsid (NC; cyan) are indicated in the RUBV cartoon, which shows virus binding to plasma membrane receptors. (A) RUBV enters the host cell by clathrin-mediated endocytosis. (B, C) In the early endosome, low pH and Ca 2+ activate virus membrane fusion. (D) NC or the genome is released into the cytoplasm. (E, F) The genomic RNA is translated to the nonstructural polyprotein p200, which synthesizes negative-strand RNAs. (G, H) p200 is processed to p150 and p90; transcription of positive polarity genomic and subgenomic RNAs then takes place. (I) The structural polyprotein precursor p110 is translated from subgenomic RNA and translocated into the ER. Signal peptidase in the ER cleaves p110 to the capsid (cyan), E2 (blue), and E1 (red) proteins. (J) The capsid is suggested to assemble with genomic RNA into NC on the RER and then to be transported to the Golgi by interactions with E2 and/or the E2-E1 dimer. (K, L) E2-E1 heterodimers are transported to the Golgi, where RUBV assembly and budding take place. (M, N) Transport vesicles deliver mature RUBV from the Golgi to the cell surface. During the process of maturation, immature particles with a uniformly dense core are believed to mature into particles with a defined internal core well separated from the virus membrane, while the smooth exterior adopts a spiky appearance For clarity, only pertinent cell organelles are diagrammed.

Recently, rubella virus has been shown to adapt to growth in granulomas present in immunocompromised vaccinees, as described in the section on vaccine reactions. The chronic replication in the cells of the granuloma results in genetic drift.

Virus isolation is generally performed in primary African green monkey kidney (AGMK) cell culture in which virus growth is detected by “challenging” the cultures with a cytopathogenic agent such as echovirus 11. If rubella virus has infected the cultures, replication of echovirus 11 is blocked, probably by interferon induction; the presence of rubella virus is inferred by this interference. Confirmation is performed by another technique, such as neutralization or fluorescence with specific antirubella serum.

Pathogenesis as It Relates to Prevention

The pathogenesis of acquired rubella infection provides two points at which immune intervention could have an effect. The first point is in the nasopharynx, where the virus first replicates and from which it spreads to local lymph nodes. Secretory immunoglobulin (Ig) A antibody in the nasopharynx, induced by prior disease or vaccination, can block mucosal replication. The second point begins about a week into the incubation period, at which time the viremia can be blocked by the presence of antibody, either passively or actively acquired.

During viremia in a pregnant woman, the virus often infects the placenta. Placental replication appears to precede fetal infections, leading to entrance of virus into the fetal circulation, from which it infects fetal organs. In vitro experiments show that human embryonic cells of many different lineages are susceptible to viral replication and develop chronic noncytopathic infection. Human stem cells differentiate less well after rubella virus infection. The same phenomenon occurs in vivo , except that only a few cells are infected at any one time. If the infected cells are stimulated to divide, either artificially in vitro or in the course of embryologic development in vivo , there is an inhibition of mitosis , that may be mediated in part by a soluble protein inhibitor or by the induction of apoptosis. Although low-passage strains produce more pronounced apoptosis, the effect is only moderate in human fibroblasts in culture. In a few organs, including the lens, cochlea, and brain, the damage caused by the virus is more cytopathic. Viral infection of the ciliary body may contribute to cataractogenesis and wild virus destruction of endothelial cells may lead to vasculitis and ischemia. Cytologic studies show that the cell skeleton and mitochondria both are damaged by rubella virus replication. Atreya and coworkers proposed that rubella virus replicase interacts with retinoblastoma tumor suppressor protein and the enzyme citron-K kinase, leading to cell-cycle arrest in S phase and activation of apoptosis. Although studies have shown that rubella infection inhibits cell division and apoptosis in infected cells, Adamo and coworkers emphasized the importance of the innate immune response to rubella infection, leading to interferon induction that causes apoptosis.

In summary, the action of rubella virus on organogenesis is mediated by varying combinations of intracellular pathology, inhibition of cellular replication, and apoptosis. ,

Diagnosis

Methods for the diagnosis of acquired and congenital rubella have been reviewed and are summarized in Table 54.5 . Clinical diagnosis of acquired infection is so inaccurate as to be useless without laboratory support. When suspected measles cases are subjected to laboratory tests, rubella infection is often identified. Isolation of virus can be accomplished from the blood and nasopharynx during the prodromal period and from the nasopharynx for as long as 2 weeks after eruption, although the likelihood of virus recovery is sharply reduced by 4 days after the rash. The Vero cell line, primary AGMK cells, or the RK13 cell line are generally used for virus isolation; but because of the slow growth of the virus in tissue culture, virus isolation is often bypassed in favor of serologic diagnosis. Isolation of virus is more common from throat swabs than from serum or plasma.

TABLE 54.5
Laboratory Diagnosis of Acquired and Congenital Rubella
Test When Positive
Specimen Acquired Rubella Congenital Rubella
Virus isolation Throat, urine, blood First week of illness At birth, declining thereafter
RT-PCR Amniotic fluid, placenta NA Throughout pregnancy
IgM antibody Serum Up to 2 months postillness At birth and first year of life
IgG antibody rise Sera Fourfold increase between acute and convalescent NA
Low IgG antibody avidity Serum Up to 2 months postillness At birth and years later
IgG antibody persistence Serum NA Beyond 6 months of age, until exposure to infection or vaccination
IgG, immunoglobulin G; IgM, immunoglobulin M; NA, not applicable; RT-PCR, reverse transcription–polymerase chain reaction.

The PCR has been adapted to the detection of rubella RNA by reverse transcription and amplification. The method appears to be 100% sensitive and approximately 90% specific and is particularly useful for prenatal detection of rubella infection of the fetus (see below). Reverse transcription–PCR was shown to permit diagnosis of rubella during the rash before appearance of IgM antibodies. The Taq Man real-time PCR assay has been applied to rubella virus detection.

Initially, serologic diagnosis became feasible because rubella virus hemagglutinates red blood cells, particularly of avian origin, permitting measurement of antibodies by hemagglutination-inhibition (HI) testing. Because HI and neutralization tests are labor intensive, other serologic examinations that are amenable to mass testing have come into use, such as latex agglutination, indirect hemagglutination, enzyme-linked immunosorbent assay (ELISA), single radial hemolysis, and fluorescence inhibition. , Any of these methods may be used to measure antibodies to rubella. Detection of antibodies in saliva or urine could simplify testing in developing countries, but commercial kits are not available. Nevertheless, identification of IgM antibody in oral or crevicular fluid permitted efficient diagnosis of rubella in the United Kingdom over a 10-year period.

Serologic diagnosis depends on the demonstration of a fourfold rise in IgG titer between acute and convalescent specimens or a demonstration of IgM antibody in the acute specimen. For IgM testing, ELISA, in a capture format to avoid false positivity because of IgG antibody, is the predominant assay in use; results may be positive for up to 6 weeks after the acute infection.

Susceptibility to rubella infection has been defined as an HI antibody titer of less than 1:8, a hemolysis-in-gel result of less than 10 IU, or an optical density by ELISA at less than the limit set by the manufacturer. However, women may lose detectable antibody and T cell proliferation in the presence of rubella antigens leading to suspicion of susceptibility but yet have protective recall responses when exposed. However, negative immunoblots identify women who are truly susceptible. Waning antibody may be restimulated on exposure, such that protection results, and epidemics have not occurred in well-vaccinated countries even when significant numbers of vaccinees no longer have antibodies at the 10 IU level. Titers at the borderline level are difficult to evaluate, but the consensus is that, although they reflect immunity in most cases, the safest response is to vaccinate the individual with equivocal levels of antibody.

Assays for antibody avidity and for responses to specific proteins or peptides have come into use for diagnosis of recent infection. , Low-avidity antibodies indicate recent infection, with maturation to high avidity by about 2 months post exanthem. A careful study of antibody responses to primary rubella infection found an initial low-avidity IgM response, followed by low-avidity IgG 3 and IgA responses, and then, finally, IgG 1 responses maturing from low to high avidity. Liebert and coworkers studied various serologic modalities and found that low levels of antibody to the major neutralizing epitope on the E1 protein, poor response to the E2 protein, and low IgG avidity for rubella virus were highly predictive of recent infection in both mother and infant. , Antibodies to E2 develop months after infection or vaccination. Rules for diagnosis of rubella in pregnancy have been well summarized.

In contrast to the situation with acquired infection, the recognition of the combination of cataracts, heart disease, and deafness provides a clinical means of diagnosis of CRS that is reasonably accurate. However, laboratory confirmation is always desirable, both because isolated abnormalities may occur and for public health reasons.

Congenital rubella infection in the infant is associated with the detection of virus, viral genome, IgM antibodies by capture techniques, low-avidity IgG antibodies, lack of antibodies to the E1 protein, or antibodies persisting beyond the predicted decay of passively transmitted maternal antibodies. Virus often can be isolated from tissues obtained at biopsy or autopsy or during surgical procedures such as cataract extraction, but more often nasopharyngeal swabs, urine specimens, or cerebrospinal fluid serve as the sources. Almost always, one or more of these sources are positive for virus at birth, gradually becoming negative during the first year of life. In severe cases, virus excretion may persist for several years. A negative result from PCR performed on fetal samples or amniotic fluid during pregnancy is good evidence against intrauterine rubella.

IgM antibodies are present in the congenitally infected infant for as long as a year after birth; however, the IgM titers decline over 6 months, whereas low-avidity IgG antibodies may persist for longer periods. Persistence of IgG antibodies beyond 6 months of age can be detected in 95% of infants with CRS. Salivary specimens are also useful in CRS for detection of antibodies. An infant with seropositive results for rubella after 6 months of age and who has not received rubella vaccine is likely to have been congenitally infected. However, CRS patients may lose their antibodies over time: 60 years after fetal infection, 40% were seronegative. Moreover, sensitization to proliferation of lymphocytes after stimulation with rubella antigens is often negative in CRS infants, even when they are seropositive, and this test may be used for diagnosis in children younger than 3 years of age. Congenital rubella can also be diagnosed by PCR tests of lens aspirates in cases of congenital cataracts.

Diagnosis of CRS has been focused on the first year of life when IgM antibody and viral shedding are likely to be present. However, a recent study of school-age children with CRS identified several serologic markers of fetal infection, including higher rubella-specific IgG and higher relative concentrations of antibodies to the core C and E2 proteins of the virus, but lower relative concentration of antibodies to the E1 protein compared with normal children. Using those markers, CRS could be identified retrospectively with 65% sensitivity and 100% specificity. Reinfection with rubella virus has been documented to rarely cause CRS, in which case IgG3 antibody is usually absent, in contrast to CRS after primary infection.

EPIDEMIOLOGY

Until 2009, rubella was a worldwide infection, as may be inferred from serologic surveys conducted in many different countries. , Before 2009, the Global Measles and Rubella Laboratory Network found rubella to be the cause of 15% of rash disease for which they received specimens. With the successful implementation of the rubella vaccination strategies in the Region of the Americas, endemic rubella and CRS in the Region of the Americas was eliminated in 2009, as verified in 2015 by the International Expert Committee on the Documentation of Elimination of Measles and Rubella. , The epidemiology of rubella as reported to WHO in 2020 showed a decrease in incidence from 13.9 cases per million in 2007 to 1.2 per million in 2018.

The factors that give rubella its epidemiologic characteristics are respiratory spread that is greater in crowded societies, different ages of infection in different epidemiological settings, and periodic disappearance from geographic areas, only to reappear in epidemic form when susceptibles accumulate. As is true of other diseases, some individual rubella patients excrete large amounts of virus in respiratory secretions and are highly infectious “spreaders,” the basic reproductive rate, Ro, was estimated to be 6–7 in developed and most developing countries but up to 12 in crowded developing countries. , In Europe, there was wide variation in the contagiousness of rubella infection between countries, with reproductive rates ranging from 3 to 8. In Mexico before vaccination, Ro varied among districts from 3.4 to 9.6, and the mean age of infection from ranged 3 to 10 years. In the World Health Organization (WHO) African Region, there was wide variation among countries in the contagiousness of infection, ranging from 3.3 to 7.9.

Many rubella infections in childhood are asymptomatic or without rash and therefore unrecognized. Daycare centers also promote early infection with rubella. In island countries and in countries that are less crowded, however, the average age at rubella infection is older, and many children reach puberty still in a seronegative state. Under these circumstances, introduction of the virus into places where young people congregate results in epidemic spread. Thus schools, colleges, and military camps are all places where rubella is likely to become epidemic. Other sites where outbreaks have also been reported include African refugee camps. Outbreaks at the stock exchange on Wall Street in the United States illustrated the potential effect of bringing seronegative individuals together in close quarters. In fact, rubella is highly efficient at infecting susceptible persons in certain epidemiologic situations.

In the United States, the epidemiology of rubella before vaccination was both endemic and epidemic. Rubella tended to occur each spring, primarily in schoolchildren 6–10 years of age, but also in older individuals. Superimposed on this occurrence was a cycle of major epidemics at 7-year intervals. Susceptibility in young adults varied from 10% to 20%, with the lower figure being found after an epidemic. Similarly, in Israel, approximately 9% of women were infected during a 1972 rubella outbreak ; however, even after the epidemic, a significant pool of susceptible individuals remained. A seroprevalence study of healthcare workers in Japan found 9.6% without rubella antibodies. Although vaccination has sharply reduced rubella in Japan, immunity is incomplete, and epidemics still occur.

The epidemiology of rubella can be deduced from the seroprevalence of rubella antibodies, including a recent review of studies done over 16 years. The large variation seen in seroprevalence suggests that rubella occurs in sporadic epidemics except where population density is high. In the metropolis of Saõ Paulo, Brazil, nearly everyone is seropositive by 20 years of age, whereas in rural Mexico, seropositivity in women of childbeaing age varies from 29% to 76%. Cutts and colleagues reviewed rubella susceptibility data from 45 developing countries and found remarkable differences among them, not correlated with geography. For example, Malaysia, Peru, and Nigeria were among the countries where more than 25% of women were found to be seronegative. Studies done in Lagos and Ibadan, Nigeria, in Thailand, and in Izmir, Turkey, demonstrated a low seroprevalence in women of childbearing age in those settings. Studies of rubella outbreaks in Kenya and Zimbabwe showed that rubella is endemic in those countries, and a serological survey in Cambodia showed only 73% seropositivity in women. In Bangladesh, the incidence of CRS was 1 per thousand births using serosurveys. Thus, even in countries where little has been done in the way of vaccination and where low socioeconomic levels would predict early transmission and high seropositivity, 5% to 25% susceptibility may be seen in women of childbearing age. Studies in various African countries have revealed ubiquitous rubella infection. For example, in South Africa, 20% of pregnant women are still seronegative. Based on these studies, the median age of infection in Africa is 7 years. In Shenzhen, China, 22% of female migrant factory workers were seronegative.

Most information on the epidemiology of CRS is available from the United States, the United Kingdom, and Europe. Table 54.6 presents the tally of fetal damage caused by rubella as a result of the 1963–1964 outbreak in the United States. At least 30,000 fetuses were damaged by intrauterine rubella, for an incidence rate of 100 per 10,000 pregnancies. In Philadelphia, the rate was also as high as 1% of pregnancies. After that outbreak, CRS rates fell to four to eight per 10,000 pregnancies until 1970, when the first vaccines were licensed. Since then, CRS declined further to an incidence of less than 0.01 per 10,000 pregnancies, with no indigenous cases, , and, with verification of elimination in the Americas, endemic rubella virus is no longer circulating. Table 54.7 gives the current surveillance criteria for CRS in the United States. In the prevaccine era, the epidemiology of CRS in the United Kingdom was similar to that in the United States, with rates of approximately 4.6 per 10,000 births. The UK has also eliminated indigenous rubella and CRS. In Europe, by the end of 2020, rubella and CRS had been eliminated in 49 of the 53 countries in the WHO European Region. , However, in 2013, observations in Italy showed that CRS was still a problem, and a large outbreak of rubella occurred in Poland. , As with other vaccine-preventable diseases, rubella outbreaks and CRS have been associated with religious minorities that refuse vaccination. An epidemic of rubella among the Pennsylvania Amish in the United States resulted in a CRS rate of more than 20 per 1,000 live births. An orthodox Protestant community in The Netherlands was the site of a large outbreak that spread to coreligionists in Canada, leading to 14 cases of CRS and two fetal deaths. Of the reported cases, 97% occurred in unvaccinated persons, but those vaccinated exhibited 99% protection.

TABLE 54.6
Estimated Morbidity Associated With the 1963–1964 Rubella Epidemic
Clinical Events Cases
Rubella cases 12,500,000
Arthritis-arthralgia 159,375
Encephalitis 2,084
Deaths
Excess neonatal deaths 2,100
Other deaths 60
Total deaths 2,160
Excess fetal wastage 6,250
Congenital rubella syndrome
Deaf children 8,055
Deaf-blind children 3,580
Mentally disabled children 1,790
Other congenital rubella syndrome 6,575
Total congenital rubella syndrome 20,000
Therapeutic abortions 5,000
From National Communicable Disease Center. Rubella Surveillance . Bethesda, MD: U.S. Department of Health, Education, and Welfare; 1969 .

TABLE 54.7
Clinical Description and Criteria for Congenital Rubella Syndrome Case Definition for the United States
Definition Case Classification
Clinical case definition: an illness, usually manifesting in infancy, resulting from rubella infection in utero and characterized by signs or symptoms from the following categories: Suspected: a case with some compatible clinical findings but not meeting the criteria for a probable case
Probable: a case that is not laboratory confirmed and that has any two complications listed in first paragraph of the clinical case definition or one complication from the first paragraph and one from the second paragraph, and lacks evidence of any other etiology
Confirmed: a clinically consistent case that is laboratory confirmed
Infection only: a case that demonstrates laboratory evidence of infection, but without any clinical symptoms or signs
•Cataracts and congenital glaucoma, congenital heart disease (most commonly patent ductus arteriosus or peripheral pulmonary artery stenosis), hearing impairment, pigmentary retinopathy
•Purpura, hepatosplenomegaly, jaundice, microcephaly, developmental delay, meningoencephalitis, radiolucent bone disease
Clinical description: presence of any defect(s) or laboratory data consistent with congenital rubella infection. Infants with CRS usually present with more than one sign or symptom consistent with congenital rubella infection. However, infants may present with a single defect. Hearing impairment is the most common single defect
Laboratory criteria for diagnosis:
•Isolation of rubella virus
•Demonstration of rubella-specific IgM antibody
•Infant rubella antibody level that persists at a higher level and for a longer period than expected from passive transfer of maternal antibody (i.e., rubella titer that does not drop at the expected rate of a twofold dilution per month)
•Detection of rubella virus by RT-PCR
CRS, congenital rubella syndrome; IgG, immunoglobulin G; IgM, immunoglobulin M; RT-PCR, reverse transcriptase–polymerase chain reaction.
From Reef SE, Plotkin S, Cordero JF, et al. Preparing for elimination of congenital rubella syndrome (CRS): summary of a workshop on CRS elimination in the United States. Clin Infect Dis . 2000;31:89 .

In recent years, the worldwide epidemiology of CRS is beginning to be known. Bozick et al. have argued that rubella frequently moves from one Asian country to another, causing large epidemics and clusters of CRS cases. In Japan, there are still many seronegative adult men, but also not all young women are seropositive. Thus, outbreaks have continued to occur. , By careful analysis of rubella on the southern Japanese island of Kyushu, Ueda and coworkers showed that the rates of both rubella and CRS were high. They concluded that the apparently low rate of CRS on Honshu is the result of a high seropositivity rate in adult women and a low clinical reporting rate of CRS. More recently, in 2012–2013 and in 2018–2019, large outbreaks of rubella occurred in Japan involving thousands of cases and many CRS babies. This led to a 2019 meeting of the Japanese Society for Vaccinology to discuss how to eliminate rubella from the country. To prevent additional rubella outbreaks, the Japanese MOH implemented a strategy of conducting a mass campaigns targeting males between 40–47 years in 2019 and those aged 48–52 years in 2020. Distributed through the local government, males in the targeted age groups were offered a coupon for free antibody testing. For those found negative, they were offered free vaccine. In China, adult women were frequently seronegative, but recent vaccination programs are leading towards rubella elimination. , Based on clinical virology studies, CRS seems to be common in Brazil. CRS is common in the developing world. , In 2011–2012, 279 CRS cases were identified in three Vietnamese hospitals. CRS cases have been identified in Nigeria, and in India, it is estimated that CRS accounts for 10% to 15% of pediatric cataracts. , Recent studies in India have demonstrated considerable incidence of congenital rubella syndrome necessitating the introduction of vaccine. In India, 26% of 90 infants investigated for congenital malformations had serologic evidence of CRS. Several other Indian studies showed that 6–25% of children with nontraumatic cataracts and 15% of infants suspected of having congenital infection had rubella-specific antibodies. A thorough retrospective study done in the Maldives gave a CRS incidence of one to four per 1,000 births during epidemics and 0.1–0.2 per 1,000 births during endemic periods. Observations made in Myanmar implied a CRS incidence of 0.1 per 1,000 births, whereas data from Bangladesh gave an incidence of 1 per 1,000 births. A history of maternal rash and fever was obtained in 40% of deaf Bangladeshi children. , An investigation in Nepal estimated a rate of CRS of 192 per 100,000 live births.

The epidemiology of rubella and CRS in Africa is incompletely described. Nevertheless, studies in Uganda, , the Democratic Republic of Congo, Nigeria, Ethiopia, Zambia, and Liberia, show that infection in children and young women is common and that congenital rubella syndrome occurs, although the precise incidence is not known. Retrospective analysis of a recent epidemic in Kumasi, Ghana gave an incidence of 0.8 per 1,000 live births. An outbreak in Tunisia was unusual because of the large number of children and adult patients with neurological symptoms. A seroprevalence study in sub-Saharan Africa showed 5% of pregnant women with rubella IgM antibodies, suggesting common infection. Although not well described, serologic data suggest that 22,500 cases of CRS are estimated to occur yearly in Africa. An incidence of 0.1 CRS cases per 1,000 live births was identified in Moroccco. A review of worldwide data concerning CRS revealed rates in developing countries varying between 0.6 and 2.2 per 1,000 live births, similar to rates seen in developed countries before universal vaccination. ,

In the absence of systematic laboratory testing of newborns, detection of cataracts and of deafness with subsequent laboratory confirmation of rubella infection is the best method for estimating the incidence of CRS. As congenital cataracts occur in approximately 25% of CRS cases, a rough estimate of incidence can be obtained by multiplying the number of patients with cataracts by four and dividing by the number of births.

CRS shows a predisposition to affect infants of young mothers, presumably because these women are more likely to enter pregnancy in a seronegative state. Women in contact with populations in which rubella outbreaks occur frequently, such as military recruits and school-age children, are more likely to be exposed. Military dependents, schoolteachers and pregnant women with older children are thus at increased risk.

Although rubella outbreaks may be explosive, they frequently do not exhaust all susceptible persons in large populations, and thus a history of having been exposed to a rubella outbreak does not necessarily indicate immunity to rubella. Even more important is the fact that 10% to 85% of infections in various outbreaks have been inapparent or at least have been without evidence of rash. Infection without rash in pregnancy can still lead to fetal disease, although the risk may be lower than that after symptomatic infection with rash.

Significance as a Public Health Problem

The seriousness of congenital rubella as a public health problem can be gauged by the results of the last major American epidemic in 1964–1965 (see Table 54.6 ). An estimated 12.5 million rubella cases occurred, including approximately 2,000 cases of encephalitis. More than 30,000 pregnancies were affected by this epidemic. Of these pregnancies, approximately 5,000 pregnant women chose to have surgical abortions, surely an underestimate, and approximately 6,250 lost fetuses to spontaneous abortions. Another 2,100 infants were stillborn or died soon after birth. CRS occurred in 20,000 infants who survived pregnancy. Of these, 11,600 were deaf, 3,580 blind, and approximately 1,800 mentally disabled. The human misery imposed by such severe damage can well be imagined. The economic burden was estimated to be US$221,660 per child with CRS, and the total cost of the epidemic may have been US$1.5 billion in 1965 dollars.

A more recent economic analysis of the impact of CRS calculated the disability-adjusted life-years and cost of care. For a CRS child in a low-income country, 29 disability-adjusted life-years and US$11,266 in cost of care would be incurred, whereas for a high-income country the figures are 19 disability-adjusted life-years and US$934,000 in cost of care. Before the use of vaccine, rubella epidemics involved approximately 5% of the population, although only approximately 10% of these cases were reported to public health authorities. In the years between epidemics, rates were about one-tenth of the epidemic peaks, but CRS continued at a low endemic rate even in those years.

Analysis of the age distribution of acquired rubella in the prevaccine era showed 60% of cases in children younger than 10 years of age and 23% in persons older than 15 years of age. As discussed subsequently, application of vaccine in children did not reduce the incidence in adolescents and adults by much, and CRS continued to occur for some years after the introduction of vaccination.

Lest it be thought that rubella infection in pregnancy has lost its danger, in 1991, a rubella outbreak among unvaccinated Amish persons living in Pennsylvania serves as a corrective. Young Amish women were 20% seronegative at the beginning of the outbreak, and infections in pregnancy were common. More than 8% of Amish infants born after the outbreak had laboratory evidence of infection, and more than 2% had CRS. Of infants born after first-trimester infection, 90% had confirmed or possible CRS.

In the United Kingdom, CRS estimates before the use of vaccine were only about 200–300 cases per year, but that figure was from a passive ascertainment system. In Australia, about one baby with CRS per 2,000 births was recorded before vaccine. During an outbreak of rubella in Israel, there were 1,441 confirmed infections in pregnant women, most of whom had abortions. Projection of the experience to the United States would be equivalent to 75,000 pregnancies complicated by rubella. In France, 16% of congenital cataract cases were attributable to CRS. A follow-up study of an epidemic in Poland showed that 15% of pregnant women had been infected. The rate of CRS after first, second, and third trimester infection was 78%, 33%, and 0%, respectively. The picture of rubella in the developing world is complex. , Although a frequent seroepidemiologic finding has been a high rate of infection early in life, there are many exceptions, including island populations, several West African countries, the city of Calcutta, and Morocco. Thus, the pattern is that of a disease that comes in epidemic waves, but that may be absent from an area for some time.

The epidemiology of CRS is known in detail for only a few countries of the world. Clusters of CRS have been reported in developing countries, , , and there is little doubt that, wherever seronegative pregnant women are exposed to a rubella outbreak, CRS cases will follow. A study conducted in Chennai, India, confirmed the presence of rubella virus in 18% of congenital cataract cases, with serologic evidence of infection in 25% (see “Epidemiology” earlier).

The most complete analysis of CRS globally was performed by Vynnycky and Adams. They modeled the incidence of the disease in all WHO member states for the years 1996 and 2010 and derived the estimate of 103,068 annual cases of CRS globally, although the confidence limits were wide, ranging from 17,146 to 269,223 cases. Table 54.8 shows their estimates.

TABLE 54.8
Estimated Incidence Rate of CRS Per 100,000 Live Births and Number of Cases of CRS by WHO Region and Globally in 1996 and 2010 a
WHO Region 1996 2010
Number Range Number Range
Africa 29,692 6,535–70,996 40,680 8,923–97,483
Americas 9,683 2,577–19,081 3 0–394
East Mediterranean 9,251 3,007–22,297 5,720 73–19,537
European 9,749 5,986–13,367 12 1–983
Southeast Asia 47,621 3,651–138,674 47,527 3,317–139,760
Western Pacific 11,707 6,617–15,676 9,127 4,831–11,066
Global 117,703 28,372–280,090 103,068 17,146–269,223

a Adams E, Vynnycky E. Unpublished data, 2012. CRS, congenital rubella syndrome; WHO, World Health Organization.

A review of rubella incidence between 2007 and 2018 concluded that the burden of rubella throughout the world has been decreasing, but, not surprisingly, the risk is greater in the countries that have yet to introduce rubella vaccine. An analysis of rubella in high and middle income countries concluded that aside from routine childhood vaccination, informing women of childbearing age about the risks of rubella in pregnancy is crucial to the induction of serological screening and demand for vaccination.

PASSIVE IMMUNIZATION

Immune Serum Globulin

Because most adults have had rubella, immune serum globulin (ISG) contains rubella antibody. By the HI test, rubella antibody titers of approximately 1:16 are found in immunoglobulins. Before the development of vaccine, ISG frequently was offered to pregnant women who had been exposed to rubella in the hope that it would prevent fetal infection. The results were equivocal, but if large doses (20–30 mL) of material were given, frank symptoms and viremia could be prevented. Experimental studies confirmed the efficacy of passive antibody in preventing both viremia and clinical rubella, , but there were numerous failures of γ-globulin to prevent congenital fetal abnormality in actual practice. , In one study of CRS patients, 6% of mothers gave histories of receipt of γ-globulin after exposure. Thus, whatever the real efficacy of ISG, it is unlikely to be complete.

The sole current indication for ISG is the exposure to rubella of a pregnant seronegative woman who will not accept abortion if infection is proved. If 1 week or less has elapsed since exposure and a serum specimen is taken first to confirm susceptibility, ISG in large amounts (20 mL) can be given intramuscularly. Convalescent specimens should be obtained 3 and 4 weeks later to search for IgM antibody and a rise in IgG titer. The absence of a rash in the exposed woman does not mean that viremia and fetal infection have been prevented. The newer intravenous γ-globulins do not have high concentrations of rubella antibodies, presumably because the antibodies are now the result of immunization rather than disease.

Hyperimmune Rubella Globulin

To overcome the deficiencies of ISG, a hyperimmunoglobulin was prepared by Cutter Laboratories (Elkhart, IN) from the sera of normal individuals who had high rubella antibody titers. The titer of rubella virus-specific antibodies in this preparation was 1:8000 by HI. In the late 1960s and early 1970s, controlled clinical trial of the preparation was performed with volunteers who were inoculated first with live unattenuated rubella virus and then given hyperimmune rubella globulin 24–96 hours later. With a challenge given intranasally, viremia was detected in two of five control subjects and in one of 10 subjects given γ-globulin. Pharyngeal excretion was similar in the two groups. Although a previous experimental study with high-titer immunoglobulin had given better results, the production of hyperimmunoglobulin was stopped, and it is not now commercially available.

ACTIVE IMMUNIZATION

Nonliving Vaccines

Advances in molecular biology allow consideration of a subunit-inactivated vaccine against rubella. The genome of the virus has been sequenced, in particular the genetic code for the E1 protein, , which carries multiple neutralizing epitopes located between amino acids 214 and 285 of the 481 amino acids contained in the polypeptide. T-cell epitopes also were defined on the E1 protein, although no single epitope was recognized by a majority of individuals. The E1 protein has been produced in quantity in baculovirus vectors and Chinese hamster ovary cells by truncating the C terminus to allow secretion. The immunogenicity of bioengineered E1 has been moderate, but strong adjuvants have produced good responses in animals. Synthetic peptides also have generated neutralizing antibodies, and virus-like particles containing the three main viral proteins have been produced from transfected cell lines, with retention of immunogenicity. , Other possible strategies for rubella vaccines include nucleic acid vaccine. Pougatcheva and coworkers raised neutralizing antibodies in mice by injecting complementary DNA coding for the envelope glycoproteins. Although it is doubtful that an inactivated vaccine could provide protection from infancy throughout the childbearing period, it might be useful for immunizing adult women.

Live Virus Vaccine

Vaccine Strains: Origin and Development

Several vaccine strains were developed soon after the isolation of rubella virus in tissue culture. Three vaccines were licensed in the United States in 1969–1970, as follows: HPV-77 (high virus passage) 77; originally passaged 77 times in monkey kidney cells and then adapted to duck embryo), HPV-77 (dog kidney), and Cendehill (rabbit kidney). In the same period, the RA27/3 human diploid fibroblast vaccine was licensed in Europe. Between 1969 and 1979, the duck embryo derivative of HPV-77 was widely used in the United States, in particular as part of the first measles-mumps-rubella (MMR) vaccine. The result was a sharp decline in rubella incidence by 1978 commensurate with vaccine coverage in children of 60% to 70%. HPV-77, in its original form and as a duck embryo–passaged virus, was immunogenic against rubella in more than 95% of subjects and, during outbreaks, gave protection to 65% to 94% of vaccinees. , Artificial challenge studies showed good protection against rubella infection. However, comparative studies of HPV-77 DE (duck embryo) versus RA27/3 conducted during the same period revealed lower antibody levels, , less persistent seropositivity, less resistance to reinfection, , less herd immunity, and more joint symptoms in those vaccinated with HPV-77, ultimately leading to its replacement by RA27/3 in a new MMR vaccine (MMR-II). HPV-77 (dog kidney) and Cendehill had been previously withdrawn from American licensure when RA27/3 was licensed in the United States in 1979, and HPV-77 (duck embryo) was then withdrawn, leaving RA27/3 as the only American rubella vaccine. The RA27/3 strain is also the exclusive vaccine strain used throughout the world, except in Japan and China ( Table 54.9 ). This strain was chosen because of its consistent immunogenicity, induction of resistance to reinfection, and low rate of side effects. Accordingly, most of the information presented hereafter concerns the RA27/3 strain, with the addition of data on the other strains that can be extrapolated to the currently used vaccine.

TABLE 54.9
Current Manufacturers of Rubella Vaccines
Manufacturer Virus Strain Cell Substrate
Merck (United States) RA27/3 HDCS
GlaxoSmithKline–RIT (Belgium) RA27/3 HDCS
Sanofi Pasteur (France) RA27/3 HDCS
Serum Institute of India RA27/3 HDCS
Kitasato Institute (Japan) Takahashi Rabbit kidney
Biken (Japan) Matsuura Quail embryo fibroblast
Takeda Chemical Industries (Japan) TO-336 Rabbit kidney
HDCS, human diploid cell strain.
Modified from Perkins FT. Licensed vaccines. Rev Infect Dis . 1985;7:S73–S76 .

RA27/3 was isolated from a fetus infected with rubella in early 1965. , Culture fluid from a tissue explant was passaged directly into WI-38 cells, and eight serial passages were made in WI-38 cultures incubated at 37°C. Additional passages then were done in cultures incubated at 30°C. After seven passages at 30°C, studies using human volunteers showed that the strain was attenuated. To reduce the pathogenicity even further, 10 additional passages were made. The RA27/3 strain is produced as a vaccine strain between the 25th and 33rd passages in human diploid cells (WI-38 or MRC-5). The relatively rapid attenuation by passage may be attributable to the use of cold adaptation, whereas the retention of high immunogenicity may be attributable to the low number of passages required to attenuate. The nucleotide sequence of the envelope genes of RA27/3 has been sequenced, revealing 31 amino acid changes in the vaccine compared with the sequence of a wild strain. In comparison, only five changes were noted in the HPV-77-DE strain.

Strain-specific nucleotide sequences have been demonstrated in the RA27/3 strain, permitting specific identification of vaccine or wild strain in case investigations. Although some antigenic variations in vaccine and wild strains of rubella virus have been discerned with rabbit antibodies against Escherichia coli –expressed proteins, studies employing a panel of monoclonal antibodies showed no significant differences between them (see “Virology” earlier).

In Japan, five Japanese strains were attenuated by passage in the following substrates: Matsuba—AGMK, swine kidney, rabbit kidney; Matsuura—AGMK, chick embryo amniotic cavity, quail embryo fibroblasts; Takahashi—AGMK, rabbit testicle, rabbit kidney; TCRB19—AGMK, bovine kidney, rabbit kidney; and TO-336—AGMK, guinea pig kidney, rabbit kidney. As of 2011, only three of these rubella vaccine strains (Takahashi, Matsuura, and TO-336) are still being administered. One Japanese vaccine strain, TO-336, has been sequenced before and after attenuation. Nucleotide mutations were discovered at 21 sites, 13 in the genes for nonstructural proteins, five in the genes for structural proteins, and three in noncoding regions. Although these mutations gave rise to 10 amino acid changes, none could be associated with attenuation. Moreover, the mutations were different from those of two other attenuated strains (RA27/3 and Cendehill). Two papers on Japanese strains attributed temperature sensitivity to mutations in the p150 nonstructural gene. , A report on the Takahashi strain showed high immunogenicity. In China a strain called BRD-II is in use, but no details of attenuation are published in English. The RA27/3 viruses being used by Merck (Whitehouse Station, NJ) and by GlaxoSmithKline (Philadekphia, PA) as part of their MMR combination have been shown to be identical by nucleotide sequencing.

Dosage and Route of Administration

The vaccine dose of RA27/3 is required to have at least 1,000 plaque-forming units (PFU) of virus delivered subcutaneously. However, titration studies in humans showed that, in keeping with its being a live vaccine, even small subcutaneous doses (<3 PFU) of RA27/3) are immunogenic. A peculiar attribute of RA27/3, not so far demonstrated with any other rubella vaccine strain, is its immunogenicity when administered intranasally. Some studies suggested that intranasal administration might confer an advantage on the vaccinees in terms of quality of the immune response. However, the subcutaneous administration of vaccine gave similar humoral antibody with only slightly less secretory antibody than intranasal administration. , Moreover, the intranasal dose of RA27/3 required for consistent immunization is high: 10,000 PFU. Lower doses result in frequent failures, particularly in children, possibly because of the mechanics of administration. Nevertheless, under careful conditions, some workers have been able to achieve 95% seroconversion rates, only slightly inferior to those rates achieved with subcutaneous injection of RA27/3.

Mexican researchers have attempted vaccination against measles, mumps, and rubella by a small-particle aerosol, and in preliminary studies have achieved seroconversion rates indistinguishable from those obtained with subcutaneous inoculation. , A trial was also done in young adults comparing an MMR vaccine given subcutaneously or by aerosol. Seroconversion of the small percentage of seronegative subjects and geometric mean titers in the two groups were quite similar after vaccination by either route.

Another potentiak route of administration is through the skin using microneedle patches. The primary barrier to global elimination of measles and rubella diseases is low vaccination coverage, especially among the most underserved populations in resource-limited settings. In contrast to conventional MR vaccination by hypodermic injection, microneedle patches are being developed to enable MR vaccination by minimally trained personnel. Simplified supply chain, reduced need for cold chain storage, elimination of vaccine reconstitution, no sharps waste, reduced vaccine wastage, and reduced total system cost of vaccination are advantages of this approach. Preclinical work to develop a MR vaccine patch has proceeded through successful immunization studies in rodents and non-human primates. Currently, there are ongoing trials in humans.

Combination with Measles and Mumps

In most developed countries, rubella vaccination is accomplished with a triple vaccine that also contains measles and mumps vaccine viruses (MMR). The triple formulation predominantly used in the United States (MMR II; Merck Sharp & Dohme, Whitehouse Station, NJ) contains the Moraten attenuated measles virus (1,000 median tissue culture infective doses [TCID 50 ]), the Jeryl Lynn mumps virus (5,000 TCID 50 ), and the RA27/3 rubella virus (1,000 TCID 50 ). Three formulations are available in Europe and elsewhere. Priorix (GlaxoSmithKline) contains the Schwarz measles virus (1,000 TCID 50 ), the Jeryl Lynn–like mumps strain (20,000 TCID 50 ), and RA27/3 rubella virus (1,000 TCID 50 ). Immune responses appear to be equivalent between MMR-II and Priorix. Trimovax (Sanofi Pasteur, Swiftwater, PA) contains the Schwarz measles virus (1,000 TCID 50 ), the Urabe mumps virus (20,000 TCID 50 ), and the RA27/3 rubella virus (1,000 TCID 50 ). Morupar (Chiron, Emeryville, CA) contains the same three viruses as in Triamox, but the mumps virus concentration is given as at least 5,000 TCID 50 . For those who prefer not to vaccinate against mumps, a measles and rubella combination is produced by Sanofi Pasteur and by the Serum Institute of India (Mumbai), which manufactures three different rubella-containing formulations: rubella-only vaccine (Wistar RA27/3; 1,000 TCID 50 ), measles-rubella (MR) (with the addition of Edmonston Zagreb measles virus; 1,000 TCID 50 ), and MMR (Tresivac, with the addition of L-Zagreb mumps strain; 5,000 TCID 50 ). A Japanese MMR combination has been made using the Takahashi strain. In China, a strain called BRD-11 is used.

Production and Constituents of Vaccine

RA27/3 is manufactured on a human diploid cell substrate, either WI-38 or MRC-5 fetal lung fibroblasts. Cell cultures inoculated with the seed virus are incubated at 30°C. After 4–7 days of initial incubation, there is sufficient virus in the supernatant medium to harvest. Fresh medium is added, and subsequent harvests can be made every 2–3 days for several weeks. Stabilizer is added to the harvest fluids, which are frozen for later safety testing and pooling before eventual lyophilization. , The final RA27/3 vaccine is essentially free of animal serum but does contain 0.4% human albumin, 25–50 µg/mL neomycin, and, in one case (Chiron), 50 µg/mL of kanamycin. However, human albumin is being replaced with recombinants-produced albumin. The lyophilization medium varies according to the manufacturer; however, it generally contains sucrose or sorbitol, glutamic acid and other amino acids, and buffering salts. When lyophilized, the vaccine is hypertonic. Reconstitution is accomplished with preservative-free sterile distilled water (0.5–1.0 mL) according to the manufacturer’s directions, which restores the vaccine to a normal or slightly hypertonic state.

RA27/3 is produced in the United States, the United Kingdom, France, Belgium and India by the manufacturers listed in Table 54.9 . Despite minor differences in dose, antibiotic content, and other details among manufacturers, differences in vaccine efficacy or in the nature or severity of side effects have not been reported.

Stability of Vaccine

Rubella vaccine is highly stable in the frozen state at approximately −70°C or approximately −20°C. At 4°C, the viability of the virus and the potency of the vaccine also are maintained for at least 5 years. At room temperature, there is significant loss after 3 months; at 37°C, a 3-week period is sufficient to damage vaccine potency. The vaccine should be stored at 2–8°C and protected from light. The virus is labile after reconstitution and should be used within 8 hours.

Results of Vaccination

Immune Responses

Vaccination induces antibodies of both IgM and IgG classes and cellular immune responses. Secretory IgA responses are also induced.

Most studies of immunogenicity have been done by measuring HI responses, although the neutralizing responses may be more important biologically. By the HI technique, 95–100% of RA27/3 vaccinees experience seroconversion by 21–28 days after vaccination, with geometric mean antibody titers ranging from 1:30 to 1:300, depending on the method of titration. , , , , Some of the apparent failures, at least in young adults, may be explained by preexisting low levels of antibody that neutralize the vaccine virus but are detectable only by sensitive tests. An ELISA titer of 10 IU is considered to confer immunity, but even lower titers may also be protective. Indeed, a meeting in 2017 called by WHO concluded that titers lower than 10 IU could be protective. By searching for antibodies against the E1 protein of the virus using immunoblot, Picone et al. showed that women with low titers by ELISA whose sera gave a positive immunoblot were actually immune. Several direct comparative immunogenicity studies have been done with different strains. In early trials, RA27/3 compared favorably with Cendehill and HPV-77, which are no longer available. The Japanese TO-336 and the Chinese BRD-II strains compared favorably with RA27/3 in parallel studies. , A vaccination study conducted in India using MMRV showed 99–100% seroconversion to rubella after one dose. RA27/3 elicits complement-fixing and precipitating antibody titers in nearly all vaccinees. In a precipitin test system, RA27/3 was the only vaccine strain that evoked antibodies to the iota internal antigen of rubella virus. Vaccination induces antibodies predominantly binding the E1 protein as detected by immunoblot, but those antibodies mature in avidity less rapidly than after natural infection and do not reach the same level.

The induction of neutralizing antibody is particularly significant and is seen regularly and promptly after the administration of RA27/3. Fig. 54.3 and Table 54.10 present comparative data obtained in New Haven, Connecticut, and in Sweden. Immunoblot analysis confirmed the persistence of antibodies for at least 3 years to the E1 protein that bears neutralizing epitopes. Antibodies to the C protein also persisted, but antibodies to E2 were often absent. A study in vaccinated children showed persistence of antibodies for at least 10 years. A crucial property of RA27/3 is its ability to induce secretory IgA antibody in the nasopharynx, which, as discussed subsequently, may prevent reinfection with wild virus. This property makes vaccination with RA27/3 similar to natural infection, which also induces local immunity. Although secretory IgA responses are higher after intranasal vaccination, they also are induced by subcutaneous vaccination with RA27/3 because of replication of the virus in the nasopharynx. IgA antibodies were detected 5 years after vaccination with RA27/3, whereas they had disappeared by that time after vaccination with other vaccine strains.

Fig. 54.3, Comparison of neutralizing antibodies and hemagglutination-inhibiting (HI) antibodies in three groups of persons: naturally infected children, children given human papillomavirus 77 duck embryo (HPV 77 DE 5 ) vaccine, and children given RA27/3 vaccine.

TABLE 54.10
Neutralizing Antibody Response in 114 Rubella-Vaccinated Women Who Demonstrated Seroconversion With Hemagglutination-Inhibiting Antibodies
Vaccine Before Vaccination a After Vaccination
8 Weeks 2 Years
Cendehill 1/45 24/43 (56%) 27/33 (82%)
HPV-77 duck 2/29 23/29 (79%) 16/17 (94%)
RA27/3 1/40 37/39 (95%) 20/20 (100%)

a Number positive/number tested. From Grillner L. Neutralizing antibodies after rubella vaccination of newly delivered women: comparison between three vaccines. Scand J Infect Dis . 1975;7:169–172 .

Not surprisingly, vaccination, like disease, is followed by the early production of IgM-class antibodies. These antibodies reach a peak at 1 month after vaccination and last approximately l month more but may persist longer at low levels.

Cellular immune responses to vaccination have been studied, although they have no known significance to protection. A proliferation of lymphoblasts in response to rubella antigen appears 2 weeks after vaccination, without suppression of tuberculin hypersensitivity. Relatively short-lived cellular responses also were seen in other studies. Human leukocyte antigen (HLA)-restricted T-cell cytotoxicity was shown to increase after immunization. Morag and coworkers showed that cytotoxic T-lymphocyte activity was high in tonsillar lymphocytes after intranasal vaccination (with RA27/3) but was low after subcutaneous vaccination (with HPV-77DE). Unfortunately, subcutaneous administration of RA27/3 was not evaluated. An association of rubella antibody response with class II but not class I HLA antigens has been demonstrated. , Elevation of tumor necrosis factor-α (TNF-α), interleukin (IL)-4 (IL-4), and IL-10 after vaccination has been reported. A study of second-dose MMR revealed effects on the distribution of lymphocyte classes, but no functional changes.

A group from the Mayo Clinic studied single nucleotide polymorphisms (SNPs) in relation to both antibody and cellular responses to rubella revaccination and have found numerous SNPs associated with higher or lower responses, although none that eliminated responses entirely. The SNPs were located in HLA genes and cytokine receptor genes. These extensive studies have recently been summarized. Males may have somewhat higher immune responses than females. Viremia has been documented between 7 and 11 days after RA27/3 inoculation. However, the viremia is low and inconstant. Pharyngeal excretion of virus is more frequent, occurring from approximately 7–21 days after vaccination, in low titer of usually less than 10 PFU per swab. Excretion peaks on approximately the 11th day after vaccination; if properly tested, essentially all vaccinees will be shown to excrete virus from the nasopharynx. , Several groups have studied third doses of rubella vaccine. Serologic responses were high and were maintained for at least 1 year. A comprehensive review of the properties of the RA27/3 vaccine strain use recently published which concluded that is generates high immunogenicity persistent protection and good safety.

Contact Spread

In view of the excretion of rubella virus by vaccinees, considerable effort has been made to detect the spread of vaccine virus to susceptible contacts. Initially, contact studies were focused on children in institutions and on families, and no evidence for spread of vaccine virus was found. , , For example, none of 393 seronegative family members was infected by contact with RA27/3 vaccinees. Veronelli studied 347 familial contacts of HPV-77DE vaccinees and also found no evidence of spread.

Experience in studies of contact spread produced largely negative results, with the rare asymptomatic seroconversion that could not be explained fully. , In the early 1970s, many studies were conducted in contacts of HPV-77DE or Cendehill strain vaccinees, including pregnant women, marital partners, school teachers, and the general population, with little or no evidence of spread. The general lack of evidence for spread by vaccine virus may reflect the maintenance of markers of attenuation by excreted virus, as demonstrated for RA27/3.

Rubella in the Measles-Mumps-Rubella Combination

Responses to rubella as part of MMR combinations are equal to those seen after rubella vaccination as a single antigen. Table 54.11 is taken from a study by Weibel and colleagues that compared MMR formulated with RA27/3 to RA27/3 alone. The excellent responses in both groups of vaccinees have been confirmed by other investigators, , who also found RA27/3 to be superior to the earlier HPV-77DE component. Seroconversion to rubella vaccination with any of the other triple combinations (see “Immune Responses” earlier) is usually 97–98%. , Bivalent measles-rubella and mumps-rubella vaccines also produce rubella antibody levels that are equivalent to those of the monovalent vaccine. , Comparative studies of the Merck and GlaxoSmithKline triple combinations showed excellent seroconversion to rubella after both vaccines. ,

TABLE 54.11
Antibody Responses in Initially Seronegative Children Who Received Combined Measles (Moraten)-Mumps (Jeryl Lynn)-Rubella (RA27/3) or Monovalent RA27/3 Rubella Vaccine
Vaccine Antibody Responses Versus
Measles (HI) Conversion (Mumps Neutralizing) Conversion Rubella (HI) Conversion
N/Total % Geometric Mean N/Total % Geometric Mean N/Total % Geometric Mean
Combined 64/68 94 57 65/68 96 8 68/68 100 136
Monovalent 67/67 100 159
HI, hemagglutination inhibition; N, number.
From Weibel RE, Carlson AJ, Villarejos VM, et al. Clinical and laboratory studies of combined live measles, mumps, and rubella vaccines using the RA27/3 rubella virus. Proc Soc Exp Biol Med . 1980;165:323–326 .

In summary, rubella vaccines induce immune responses that are similar in quality but lesser in quantity than those after natural disease. , The live virus vaccine produces viremia and pharyngeal excretion, but both are of low magnitude and are noncommunicable. IgM and IgG antibody responses follow vaccination. Natural infection elicits nasal secretory antibody that may be useful in the prevention of reinfection, and RA27/3 vaccine also has the same property.

Effect of Maternal Antibody

On average, maternal rubella antibody levels are detected only for approximately 2 months after birth in the infant, and thus rubella vaccine does not suffer the same problem as measles vaccination in early life, where maternal antibodies interfere with infant vaccination. Studies have documented that the seroconversion rate in infants at 9 months of age is similar to that of children older than 12 months of age. , ,

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