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Investigation of mumps vaccine failures in Minsk, Belarus, 2001–2003
Vaccine 25 (2007) 4651–4658
Investigation of mumps vaccine failures in Minsk, Belarus, 2001–2003 Alena V. Atrasheuskaya a,∗ , Elena M. Blatun b , Michail V. Kulak a , Alina Atrasheuskaya c , Igor A. Karpov c , Steven Rubin d , George M. Ignatyev a a
State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region 630559, Russia b Hospital of Infectious Diseases, Minsk 220050, Belarus c Infectious Diseases Department, State Medical University, Minsk 220116, Belarus d Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892, USA Received 12 June 2006; received in revised form 3 April 2007; accepted 10 April 2007 Available online 25 April 2007
Abstract The purpose of this study was to investigate mumps vaccine failures (VF) in a highly vaccinated population of Minsk, Belarus, and to investigate a possible role for virus strain-specific immunity. During our 3-year study period, 22 adults were admitted to the Infectious Diseases Hospital in Minsk with a diagnosis of mumps. A genotype H1 mumps virus (MuV) strain was identified in all patients. Of 15 patients from whom the paired sera were collected, 9 were confirmed to have been previously vaccinated. Serological examinations indicated primary VF in seven of these cases and secondary VF in two. Despite almost all vaccinated patients possessing MuV specific IgG, few possessed neutralizing antibody to the vaccine strain and titers were nominal. Importantly, none of the sera were able to neutralize a genotype H MuV strain. Our results demonstrate the importance of assaying for neutralizing antibody and support the assertion that antigenic differences between wild type and vaccine MuV strains may play a role in cases of breakthrough infection in vaccinees. © 2007 Elsevier Ltd. All rights reserved. Keywords: Belarus; Mumps genotyping; Vaccine failure
1. Introduction Mumps is a vaccine preventable disease. Live attenuated mumps vaccines have been available since the mid 1960s. In Belarus, a mandatory single dose of mumps vaccine was instituted in 1986 and a two-dose mandatory regimen (first dose at 12–24 months of age, second dose at 6–7 years of age) was instituted in 1999. The Russian monovalent vaccine containing the Leningrad-3 (L-3) MuV strain was used until 1996, since then the “Trimovax”, trivalent measles, mumps, rubella vaccine, containing the Urabe AM-9 MuV ∗ Corresponding author at: Laboratory of Immunology Safety, State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region 630559, Russia. Tel.: +7 383 236 74 48; fax: +7 383 236 74 09. E-mail addresses: [email protected], [email protected] (A.V. Atrasheuskaya).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.04.020
strain (Aventis Pasteur, France) has been used [1]. In response to the widespread use of a two dose mumps vaccine regimen instituted in 1999, the number of notified cases of mumps in Belarus have declined significantly, from 23,104 cases in 1999 to only 759 cases in 2004 [2]. At this time there is no information on MuV genotypes circulating in the Republic. There has been some shift in the age distribution of mumps cases in Belarus toward the older age’s vaccinees [3]. The occurrence of sporadic mumps outbreaks in populations with high vaccine coverage is a well-known phenomenon. These outbreaks are usually attributed to pockets of unvaccinated individuals or to VFs, primary or secondary [4–7]. It has been suggested that antigenic differences between MuV strains may allow for certain strains to escape neutralization in vaccinees [8–10]. In the present prospective study we examined cases of mumps in adults (>14 years) admitted to the Infectious Diseases Hospital in Minsk during 2001–2003 years. This was not a controlled
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epidemiological study. Minsk is the largest city in Belarus with a population of about 1.8 million residents and with a reported 98.5% mumps vaccine coverage rate. The numbers of officially reported mumps cases in Minsk during the study years were as following: 60.9 cases/100,000 population in 2001, 42.6 cases/100,000 population in 2002 and 21.8 cases/100,000 population in 2003. For all cases of mumps in our study the vaccination status was determined. Acute and convalescent saliva and sera samples were obtained. RT-PCR testing of saliva using primers specific for the mumps virus SH gene was performed to confirm the presence of the virus as well as to identify the virus genotype [11,12]. Sera were tested by ELISA for IgM and IgG antibody titers and for IgG antibody avidity. In addition, sera were tested for ability to neutralize the vaccine strain (L-3) as well as two wild type viruses supposed to be circulating in Belarus based on the data from the neighboring countries [13–16].
2. Subjects and methods
1:231. Samples with OD values below 0.100 (negative cutoff) were regarded as negative. Samples within 0.100–0.200 OD values were retested and defined thereafter as negative, positive or equivocal. The IgG samples with OD values within 0.200–2.500 were regarded as positive and the titre dilution was calculated according to the manufacturer’s instruction. Samples with OD values higher than 2500 (positive cut-off) were diluted appropriately, and retested as recommended by manufacturer. Assessment of IgG avidity in acute sera was carried out using the above referenced IgG ELISA and 6 M urea as described earlier [18]. Avidity index (AI) was calculated as the ratio of the absorbencies with and without urea treatment. Using a previously established system, an AI of less than or equal to 31 was considered to represent low avidity while values greater than or equal to 32 were considered to represent high avidity [18]. The differential diagnosis [19] was made by testing serum in an ELISA for antibodies to parainfluenza virus types 1–3 (Parainfluenza 1/2/3 IgG-ELISA; IBL) and to Epstein-Barr virus (Enzygnost Anti-EBV/IgM; Dade Behring).
2.1. Subjects From January 2001 to December 2003 a total of 22 adults were admitting to the Infectious Diseases Hospital in Minsk (Belarus) with a diagnosis of mumps. All 22 patients presented with parotitis, were admitted to the hospital within 3 days of symptom onset, and were prospectively enrolled in our study. Hospital admission was mostly based on the need for quarantine rather than for medical treatment. Clinical diagnosis of mumps was made based on the WHO mumps case definition [17]. Institutional Review Board approval (IRB00001360) was obtained and informed consent was received from all subjects. The age of the patients ranged from 16 to 26 years. There were 6 females and 16 males. Sixteen of the patients had previously received one dose of mumps L-3 vaccine at 12–24 months of age, according to the official medical records. One patient had not been vaccinated and the vaccination status of the remaining five patients could not be confirmed. The clinical diagnosis of mumps infection was confirmed in all 22 patients by MuV-RNA detection in saliva samples by RT-PCR. The nucleotide sequences obtained from those 22 were used to genotype the viruses. For serological studies (IgM, IgG and neutralizing antibody levels), only 15 of the 22 patients were included due to the lack of paired acute and convalescent sera from 7 of the patients. 2.2. Methods 2.2.1. ELISA Serum samples were taken at hospital admission and at discharge (6–10 days later). Serological detection of antimumps virus IgM and IgG antibodies was made by the commercial ELISA (Enzygnost, Dade Behring, Marburg, Germany) according to the manufacturer’s instructions. For both IgM and IgG ELISA testing, samples were prediluted
2.2.2. Plague reduction neutralization assay Mumps virus neutralizing antibody titers were determined by plaque reduction neutralization assay (PRN) as previously described [20]. Briefly, sera were thawed at room temperature and heated at 56 ◦ C for 45 min to inactivate complement. Two-fold serial dilutions of serum (or media alone as a negative control) were mixed with equal volumes of approximately 30–40 plaque forming units (pfu) of the target virus to give a final sera dilution range of 1:4 to 1:128. Virusserum mixtures were incubated at 37 ◦ C, 5% CO2 for 1 h and then placed on Vero cell monolayers in 24-well plates and incubated for one hour at 37 ◦ C, 5% CO2 . The virus-serum mixture was removed by aspiration and cell monolayers were rinsed with minimal essential medium (MEM) immediately before covering with 0.75% agar Nobel (Sigma Chemicals, St. Louis, MO) in MEM supplemented with 10% fetal bovine serum (SRC VB “Vector”, Koltsovo, Russia). Plates were then incubated at 37 ◦ C, 5% CO2 for 5 days. A second layer of agar containing 0.01% neutral red (Quality Biologicals, Gaithersburg, MD.) was added and incubated overnight to visualize plaques produced by remaining infectious virus. All procedures were done in triplicate for each test serum. The neutralizing antibody titer was determined as the mean highest dilution of serum capable of reducing the number of virus plaques by 50% or greater compared to control values (virus incubated with negative control serum). The minimal detectable neutralizing antibody titer in this assay is 1:4 (the cut-off for seropositivity). The targets for neutralization included the L-3 vaccine mumps virus (kindly provided by the Tarasevich State Institute of Standardization and Control of Immunobiological Preparations, Moscow, Russia), a genotype H wild type strain (PetroNov, Genbank Access No. AY681495) and a genotype C wild type strain (Dragoon, Genbank Access No. AY669145). The wild-type genotype C and
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Table 1 Serum levels of mumps virus IgM, IgG titer, IgG avidity, neutralizing antibody titers of mumps patients Patient
Age
Gender
Vaccination status
IgM in acute sera
IgG (titer)a
P1/2001 P2/2001 P3/2002 P4/2002 P5/2002 P6/2002 P7/2002 P8/2002 P9/2002 P10/2002 P11/2002 P12/2003 P13/2003 P14/2003 P15/2003
21 16 22 20 18 20 19 18 22 18 19 20 26 18 18
M M F M M M M F M F M F M M M
Unknown Vaccinated 14 years ago Vaccinated 20 years ago Vaccinated 18 years ago Unknown Unknown Vaccinated 17 years ago Vaccinated 16 years ago Vaccinated 20 years ago Unknown Vaccinated 17 years ago Unknown Unvaccinated Vaccinated 14 years ago Vaccinated 16 years ago
+ + + + + + + ± ± ± + + + – +
1:3600/1:4800 1:1000/1:2600 1:1.400/1:3.600 neg/1:2400 neg/1:3200 1:2100/1:8200 1:1700/1:5900 1:3300/1:9600 1:1400/1:3000 1:2600/10700 neg/1:300 1:600/1:1300 1:200/1:1200 1:500/1:2000 1:1500/1:7500
AI in acute sera (%) 23 27 19 – – 26 45 24 19 22 – 18 31 44 17
PRN with strain L-3 (titer)a
PRN with strain H (titer)a
PRN with strain C (titer)a
1:4/1:4 1:4/1:4 n.d/1:4 n.d./1:4 n.d./1:4 n.d./1:4 1:8/1:8 n.d./1:8 n.d./1:8 n.d./1:4 n.d./1:4 1:4/1:8 1:4/1:4 1:8/1:8 n.d./1:8
n.d./1:4 n.d./1:8 n.d./1:4 n.d./1:8 n.d./1:8 n.d./1:4 n.d./1:8 n.d./1:16 n.d./1:16 n.d./1:4 n.d./1:4 n.d./1:8 n.d./1:4 n.d./1:4 n.d./1:16
n.d./n.d. n.d./1:4 n.d./n.d. n.d./1:4 n.d./1:4 n.d./1:4 n.d./n.d. n.d./1:4 n.d./1:4 n.d./1:4 n.d./n.d. n.d./1:8 n.d./1:4 n.d./n.d. n.d./1:4
All vaccinated here patients were immunized only once, as a mandatory second vaccination was introduced in Minsk in 1999. AI: avidity index; PRN: plaque reduction neutralization assay; n.d.: not detectable level. a Acute serum samples were taken at admission/second samples were taken at discharge. Paired samples were taken with the interval of 7–12 days.
H viruses were isolated in Novosibirsk, Russia [13,16]. A negative serum control was included in each plate of the tested samples. In all PRN-assays, no virus neutralizing activity was measured with the negative control sample. 2.2.3. Nucleotide sequence analysis RNA was extracted from saliva and sera and was purified using QIAGEN RNeasy mini kit (Qiagen, Germany). For identifying the specific virus strain responsible for infection, RT-PCR was performed on saliva samples using specific primers to amplify the viral SH gene as previously described [21]. The primers for RT-PCR were 5 -tcaacacaatatcaagta3 (pos. 2964–2981) and 5 -ttctgtgttgtattgtga-3 (pos. 3401–3418). The PCR products were excised from the gel and purified using QIAquick Gel Extraction Kit (Qiagen, Germany). The purified products were directly sequenced the primers 5 -atgatctcatcaggtac-3 (pos. 2988–3004) and 5 tcctaagtttgttctgg-3 (pos. 3384–3400). Nucleotide sequences were determined by the CEQ 2000 Dye Terminator Cycle Sequencing Kit (Beckman Coulter, USA) and Beckman CEQ 2000 XL DNA Analysis System (Beckman Coulter, USA) according to manufacturer’s instructions. Nucleotide alignments analyses were performed using Vector NTI v. 8.0 (InforMax, Bethesda, MD) software package. The MEGA package (version 3.1) was used to generate a phylogenetic tree using neighbor joining and Kimura two-parameter methods. The statistical significance of a particular tree topology was evaluated by bootstrap re-sampling of the sequences 1000 times. 2.2.4. Statistical analysis Mumps PRN titers were normalized by logarithmic transformation to calculate mean titers (MT). In all cases, sera that were below the level of detection (titer less than 1:4) were
arbitrarily assigned a titer of 1:2 (1 − log2 ) for the purpose of statistical analysis. Quantitative results were expressed as MT ± S.D. (standard deviation). The statistical significance between MT was tested. Statistical analysis was conducted using Student’s t- or Chi square-test. Statistical significance was defined at P ≤ 0.05. Pearson correlation coefficient (r) was also calculated. 2.3. Definitions Primary vaccine failure (PVF) was defined as a previously vaccinated subject with clinical mumps and evidence of a primary immune response (PIR): IgM positive/IgG negative or IgG positive with low avidity [22]. Secondary vaccine failure (SVF) was defined as a previously vaccinated subject with clinical mumps and evidence of a secondary immune response (SIR): high avidity IgG antibody in acute phase sera [18].
3. Results 3.1. Clinical manifestations and vaccination history Twenty-two adults were admitted to the Infectious Diseases Hospital in Minsk with a diagnosis of mumps, all presenting with parotitis. Paired serum samples were obtained from 15 of these patients. All subsequent studies described henceforth are limited to this subgroup of 15 patients. As summarized in Table 1, nine of these patients received one dose of mumps-containing vaccine 14–20 years earlier at 12–24 months of age. One patient had not been previously vaccinated and the vaccination status of the remaining five patients could not be confirmed. Ten patients had bilateral
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parotitis (67%) and five had unilateral parotitis (33%). The submaxillary salivary glands were involved in nine patients (60%). Pancreatitis was diagnosed in two patients (13.3%), P1 and P2. Orchitis/epidimoorchitis was found in three males (20%), P1, P2 and P12. Patients P7, P13 and P14 had subfebrile body temperature while others were febrile. Clinical symptoms of meningitis (headache, vomiting, and fever) were registered in five patients (33.3%), P4, P5, P6, P10, P11, however spinal taps were not performed, thus the diagnosis could not be confirmed. The duration of the clinical course of mumps infection was from 7 to 12 days. All patients recovered completely. 3.2. Serological results All patients were negative for acute EBV and parainfluenza viruses 1, 2, and 3 by ELISA. Serum levels of MuV-specific IgM and IgG titer, IgG avidity, neutralizing antibody titers are shown in Table 1. IgM was detected in 11 of the 15 patients upon hospital admission. One patient (P14) was IgM negative and the presence of IgM was equivocal in three patients (P8–P10). Three patients were IgG negative at admission (P4, P5 and P11) but seroconverted by hospital discharge. The increase in IgG titers of the remaining patients was diagnostically significant (>2.1-fold), except for patient P1, who possessed a very high initial IgG titer. There was no correlation between the appearance of IgM and IgG concentration, nor between IgM and IgG levels and vaccination status. According to the definitions of vaccine failure, of the nine patients for whom vaccination could be positively confirmed, seven were cases of PVF (P2–P4, P8, P9, P11 and P15) and 2 were cases of SVF (P7 and P14). Vaccine virus (L-3) neutralizing antibodies were only detected in three of the nine vaccinees (P2, P7 and P14) at admission. Interestingly, the L-3 neutralizing antibody titers did not rise by discharge for these three patients. Vaccine virus neutralizing titers were in general higher at discharge than at admission (1.5 ± 0.7 − log2 dilution versus 2.4 ± 0.5 − log2 dilution; P < 0.0009). There was a good correlation between the AIs and the neutralizing antibody titers against the L-3 vaccine strain in the acute sera, r = 0.71, P < 0.05. None of the patients, including those for whom vaccination status could not be confirmed, had detectable neutralizing antibody against a wild type mumps genotype H strain upon admission. By hospital discharge, all 15 patients had neutralizing antibody against this strain. Notably, a strain similar to the one used here was detected in saliva of all patients by RT-PCR. Mean neutralizing titers against the genotype H virus were greater than those against a genotype C virus strain also tested in this study (2.7 ± 0.8 − log2 dilution versus 1.7 ± 0.6 − log2 dilution, respectively; P < 0.0006). The correlation between the neutralizing titers against mumps virus genotypes H and C in patients at discharge was moderate (r = 0.53; P < 0.05).
Patients P7, P13 and P14 had the highest avidity indexes and neutralizing titers and had the mildest clinical course of mumps infection. Those with symptoms of meningitis (P4, P5, P6, P10 and P11), had no detectable neutralizing antibody titers at admission and tended to have the lowest IgG AI scores (14.8 ± 3.8 versus 26.7 ± 3.3, P < 0.05), and three of those five (P4, P5 and P11) were initially IgG negative. 3.3. Nucleotide sequence analysis The diagnosis of mumps in all 22 patients admitted to the hospital was confirmed by RT-PCR. However, only the acute patients’ saliva samples were RT-PCR positive, but not serum samples. In all cases, the amplified SH gene sequence was identical and belonged to genotype H, based on the established genotype classification system for mumps viruses [23]. RNA extraction and RT-PCR studies were done in different rooms using air barrier filter pipette tips, so contamination and false positive results are highly unlikely. The precise sequence of this wild type H virus was deposited in Genbank under accession numbers (one per year) DQ136174 (P1/2001), DQ136175 (P4/2002) and DQ250041 (P14/2003), respectively. A phylogenetic comparison of the SH gene sequence of the different genotype H MuV isolates around the world are shown in Fig. 1. Notably, the isolates obtained in Belarus differed from the H2 strain isolated from nearby Russia (PetroNov-Russia-2004). The genotype H clinical isolates obtained from patients in the present study were most similar to the H1 subgroup (Fig. 1) reported earlier [23]. However, all Belarusian sequences contained an L at position 30 instead of the S typical to the H1 group [23]. In addition, all isolates contained an S instead of P at position 6. The latter was not previously reported for genotype H viruses [24]. Thus, the Belarusian isolates formed a distinct cluster within the genotype H1 group.
4. Discussion Vaccine failures have been linked to mumps outbreaks in populations with high vaccine coverage [13–16,25]. The assessment of VF cases provides information useful for the improvement of mumps immunization strategy. The WHO strategies to achieve mumps elimination include not only very high coverage with the first dose of mumps vaccine, but also a second opportunity for vaccination and conducting catchup immunization of susceptible cohorts [17]. It is well known that VFs are less common in recipients of a two-dose regimen [14,26,27]. In all cases of mumps vaccine failure in our study, only one dose of vaccine was administered, and in most of the cases primary VF (7 from 9 vaccinated patients) was identified based on the AIs. It should be mentioned that different concentrations of urea used in mumps virus IgG
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Fig. 1. Genotype C sequences were included as an out-group. Phylogenetic analysis was performed using the Kimura two-parameter model as a model of nucleotide substitution and using the neighbor-joining method to reconstruct the phylogenetic tree (MEGA version 3.1 software package). The statistical significance of the phylogenies constructed was estimated by bootstrap analysis with 1000 pseudoreplicate data sets.
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avidity assay might affect assessment of secondary vaccine failure rate in outbreak settings [18,25]. Wherein, low avidity IgG signifies an immune response in immunologically naive patients, high avidity IgG signifies an immune response in patients with preexisting B-cell memory [28]. Notably, in contrast to conventional thinking, IgM can be detected in secondary immune responses (as we found in P7 here) and therefore does not serve as a marker of primary infection per se [25,29,30]. In many of our cases, patients possessed high mumps virus specific ELISA IgG titers in acute phase sera, yet avidity was low. The lack (or weak) association between IgG titers and their avidity have been reported earlier for a number of agents [31–34]. The PRN assay using the L-3 vaccine strain was invaluably helpful in the assessment of VF cases in our study. With one exception, all of the vaccinated patients assigned to the PVF-group lacked antibody neutralizing activity against the L-3 mumps vaccine strain despite being IgG-positive. At the same time, both patients assigned to the SVF-group demonstrated a pretty mature immune response based on the AIs as 44–45% which was also characterized by the PRN titer against L-3 vaccine strain as 1:8. Interestingly, at discharge the majority of patients demonstrated a rise in neutralizing titers against the L-3 strain independently of their vaccination history. The main finding here was that all patients independent of vaccination history, lacked neutralizing antibodies against the genotype H MuV, which is likely responsible for their susceptibility. In a recent publication, data was presented supporting the notion that sera possessing only low levels of mumps neutralizing antibody may not be broadly protective due to antigenic differences between MuV strains [35]. This is in line with reports by others proposing genotypespecific antibody responses [36,37]. In the present study, all acute phase sera possessing neutralizing antibody were of low titer and were capable of neutralizing the L-3 vaccine virus strain but not the wild type genotype H or C MuV strains tested. However, at discharge all sera demonstrated a MuV-specific neutralizing antibody response in that all convalescent sera could more effective neutralize a genotype MuV strain similar to the one detected by RT-PCR in patient saliva in comparison with a contemporary wild genotype MuV strain. This highlights the need for a more intensive examination of levels of neutralizing antibody required to confer broad cross-strain protection. Considering that mumps virus is serologically monotypic (despite antigenic difference between virus strains), such a “threshold” titer should exist. Our data indicate the importance of the assessment of avidity index and neutralizing antibody titers, against both the vaccine virus and the circulating viruses, for investigation of VF cases. The simplified dichotomic classification of vaccine failures (primary versus secondary) based only on the AIs may be problematic since a “grey zone” likely exists as was noted for measles by Paunio [38]. Earlier Narita et al. [39] also speculated that SVF comprises a wide variety of immune
responses to vaccine and that one extreme mimics PVF in terms of not only clinical presentation, but also serological response. The latter could take place in some of our patients. The PVF-group formed on the basis of AIs in our study consisted of seronegative subjects as well as seropositive subjects possessing IgG of low AIs, either lacking neutralizing antibody or possessing minimal titers of neutralizing antibody. Moreover, basing VF only on antibody data may be misleading and incomplete [40,41]. The reflexive focus on humoral immunity has overshadowed the role of the cellular response. Some of the mumps VF cases in our patients may be more clearly explained by the additional investigation of cellular mechanisms. For measles, individuals with undetectable or low levels of antibody may not necessarily be susceptible to infection, since cell-mediated immunity also affords protection [40,41]. Thus, it is of importance to develop a reliable laboratory protocol specifying the tests for investigation both cellular and humoral immune responses. The latter could shed much needed light on the issue of the VF and its classification. Genetic analysis is indispensable in understanding the epidemiology of mumps infection and in examining possible genotype-specific immunity in the population. Interestingly, the Belarus isolates belonged to a distinct cluster within the genotype H1 group of MuV. That this particular strain was isolated from all patients in our study over the 3-year period suggests that this strain is endemic to this geographic region. In conclusion, in all cases of vaccine failure identified in our study, only one dose of vaccine was administered and in most of these cases, primary VF was implicated based on the absence of markers suggestive of a mature and functional immune response. Whether or not the absence of sufficient titers of neutralizing antibody in response to vaccination or the absence of neutralizing antibodies specific for the endemic mumps virus strain were responsible for susceptibility of vaccinees to infection cannot be conclusively determined, but both are indicated. What is clear is that cause of vaccine failure is complex and multifactorial. It is therefore of high importance to develop a reliable laboratory protocol specifying all tests to be performed, analyzed and interpreted for VF case investigation. Our findings suggest that proper assessment of vaccine failure should include IgG avidity testing as well as a comprehensive assessment of the functional antibody response, i.e., the differential ability of serum antibody to neutralize both the vaccine virus as well as the endemic challenge virus strain.
Acknowledgements The work has been done in the framework of a grant BTEP ID#16, 2002. No official support or endorsement of this article by the Food and Drug Administration is intended or should be inferred. We thank our colleges Malisheva T. and D’achkova L. for the excellent technical assistance.
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Investigation of mumps vaccine failures in Minsk, Belarus, 2001–2003 Alena V. Atrasheuskaya a,∗ , Elena M. Blatun b , Michail V. Kulak a , Alina Atrasheuskaya c , Igor A. Karpov c , Steven Rubin d , George M. Ignatyev a a
State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region 630559, Russia b Hospital of Infectious Diseases, Minsk 220050, Belarus c Infectious Diseases Department, State Medical University, Minsk 220116, Belarus d Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892, USA Received 12 June 2006; received in revised form 3 April 2007; accepted 10 April 2007 Available online 25 April 2007
Abstract The purpose of this study was to investigate mumps vaccine failures (VF) in a highly vaccinated population of Minsk, Belarus, and to investigate a possible role for virus strain-specific immunity. During our 3-year study period, 22 adults were admitted to the Infectious Diseases Hospital in Minsk with a diagnosis of mumps. A genotype H1 mumps virus (MuV) strain was identified in all patients. Of 15 patients from whom the paired sera were collected, 9 were confirmed to have been previously vaccinated. Serological examinations indicated primary VF in seven of these cases and secondary VF in two. Despite almost all vaccinated patients possessing MuV specific IgG, few possessed neutralizing antibody to the vaccine strain and titers were nominal. Importantly, none of the sera were able to neutralize a genotype H MuV strain. Our results demonstrate the importance of assaying for neutralizing antibody and support the assertion that antigenic differences between wild type and vaccine MuV strains may play a role in cases of breakthrough infection in vaccinees. © 2007 Elsevier Ltd. All rights reserved. Keywords: Belarus; Mumps genotyping; Vaccine failure
1. Introduction Mumps is a vaccine preventable disease. Live attenuated mumps vaccines have been available since the mid 1960s. In Belarus, a mandatory single dose of mumps vaccine was instituted in 1986 and a two-dose mandatory regimen (first dose at 12–24 months of age, second dose at 6–7 years of age) was instituted in 1999. The Russian monovalent vaccine containing the Leningrad-3 (L-3) MuV strain was used until 1996, since then the “Trimovax”, trivalent measles, mumps, rubella vaccine, containing the Urabe AM-9 MuV ∗ Corresponding author at: Laboratory of Immunology Safety, State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region 630559, Russia. Tel.: +7 383 236 74 48; fax: +7 383 236 74 09. E-mail addresses: [email protected], [email protected] (A.V. Atrasheuskaya).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.04.020
strain (Aventis Pasteur, France) has been used [1]. In response to the widespread use of a two dose mumps vaccine regimen instituted in 1999, the number of notified cases of mumps in Belarus have declined significantly, from 23,104 cases in 1999 to only 759 cases in 2004 [2]. At this time there is no information on MuV genotypes circulating in the Republic. There has been some shift in the age distribution of mumps cases in Belarus toward the older age’s vaccinees [3]. The occurrence of sporadic mumps outbreaks in populations with high vaccine coverage is a well-known phenomenon. These outbreaks are usually attributed to pockets of unvaccinated individuals or to VFs, primary or secondary [4–7]. It has been suggested that antigenic differences between MuV strains may allow for certain strains to escape neutralization in vaccinees [8–10]. In the present prospective study we examined cases of mumps in adults (>14 years) admitted to the Infectious Diseases Hospital in Minsk during 2001–2003 years. This was not a controlled
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epidemiological study. Minsk is the largest city in Belarus with a population of about 1.8 million residents and with a reported 98.5% mumps vaccine coverage rate. The numbers of officially reported mumps cases in Minsk during the study years were as following: 60.9 cases/100,000 population in 2001, 42.6 cases/100,000 population in 2002 and 21.8 cases/100,000 population in 2003. For all cases of mumps in our study the vaccination status was determined. Acute and convalescent saliva and sera samples were obtained. RT-PCR testing of saliva using primers specific for the mumps virus SH gene was performed to confirm the presence of the virus as well as to identify the virus genotype [11,12]. Sera were tested by ELISA for IgM and IgG antibody titers and for IgG antibody avidity. In addition, sera were tested for ability to neutralize the vaccine strain (L-3) as well as two wild type viruses supposed to be circulating in Belarus based on the data from the neighboring countries [13–16].
2. Subjects and methods
1:231. Samples with OD values below 0.100 (negative cutoff) were regarded as negative. Samples within 0.100–0.200 OD values were retested and defined thereafter as negative, positive or equivocal. The IgG samples with OD values within 0.200–2.500 were regarded as positive and the titre dilution was calculated according to the manufacturer’s instruction. Samples with OD values higher than 2500 (positive cut-off) were diluted appropriately, and retested as recommended by manufacturer. Assessment of IgG avidity in acute sera was carried out using the above referenced IgG ELISA and 6 M urea as described earlier [18]. Avidity index (AI) was calculated as the ratio of the absorbencies with and without urea treatment. Using a previously established system, an AI of less than or equal to 31 was considered to represent low avidity while values greater than or equal to 32 were considered to represent high avidity [18]. The differential diagnosis [19] was made by testing serum in an ELISA for antibodies to parainfluenza virus types 1–3 (Parainfluenza 1/2/3 IgG-ELISA; IBL) and to Epstein-Barr virus (Enzygnost Anti-EBV/IgM; Dade Behring).
2.1. Subjects From January 2001 to December 2003 a total of 22 adults were admitting to the Infectious Diseases Hospital in Minsk (Belarus) with a diagnosis of mumps. All 22 patients presented with parotitis, were admitted to the hospital within 3 days of symptom onset, and were prospectively enrolled in our study. Hospital admission was mostly based on the need for quarantine rather than for medical treatment. Clinical diagnosis of mumps was made based on the WHO mumps case definition [17]. Institutional Review Board approval (IRB00001360) was obtained and informed consent was received from all subjects. The age of the patients ranged from 16 to 26 years. There were 6 females and 16 males. Sixteen of the patients had previously received one dose of mumps L-3 vaccine at 12–24 months of age, according to the official medical records. One patient had not been vaccinated and the vaccination status of the remaining five patients could not be confirmed. The clinical diagnosis of mumps infection was confirmed in all 22 patients by MuV-RNA detection in saliva samples by RT-PCR. The nucleotide sequences obtained from those 22 were used to genotype the viruses. For serological studies (IgM, IgG and neutralizing antibody levels), only 15 of the 22 patients were included due to the lack of paired acute and convalescent sera from 7 of the patients. 2.2. Methods 2.2.1. ELISA Serum samples were taken at hospital admission and at discharge (6–10 days later). Serological detection of antimumps virus IgM and IgG antibodies was made by the commercial ELISA (Enzygnost, Dade Behring, Marburg, Germany) according to the manufacturer’s instructions. For both IgM and IgG ELISA testing, samples were prediluted
2.2.2. Plague reduction neutralization assay Mumps virus neutralizing antibody titers were determined by plaque reduction neutralization assay (PRN) as previously described [20]. Briefly, sera were thawed at room temperature and heated at 56 ◦ C for 45 min to inactivate complement. Two-fold serial dilutions of serum (or media alone as a negative control) were mixed with equal volumes of approximately 30–40 plaque forming units (pfu) of the target virus to give a final sera dilution range of 1:4 to 1:128. Virusserum mixtures were incubated at 37 ◦ C, 5% CO2 for 1 h and then placed on Vero cell monolayers in 24-well plates and incubated for one hour at 37 ◦ C, 5% CO2 . The virus-serum mixture was removed by aspiration and cell monolayers were rinsed with minimal essential medium (MEM) immediately before covering with 0.75% agar Nobel (Sigma Chemicals, St. Louis, MO) in MEM supplemented with 10% fetal bovine serum (SRC VB “Vector”, Koltsovo, Russia). Plates were then incubated at 37 ◦ C, 5% CO2 for 5 days. A second layer of agar containing 0.01% neutral red (Quality Biologicals, Gaithersburg, MD.) was added and incubated overnight to visualize plaques produced by remaining infectious virus. All procedures were done in triplicate for each test serum. The neutralizing antibody titer was determined as the mean highest dilution of serum capable of reducing the number of virus plaques by 50% or greater compared to control values (virus incubated with negative control serum). The minimal detectable neutralizing antibody titer in this assay is 1:4 (the cut-off for seropositivity). The targets for neutralization included the L-3 vaccine mumps virus (kindly provided by the Tarasevich State Institute of Standardization and Control of Immunobiological Preparations, Moscow, Russia), a genotype H wild type strain (PetroNov, Genbank Access No. AY681495) and a genotype C wild type strain (Dragoon, Genbank Access No. AY669145). The wild-type genotype C and
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Table 1 Serum levels of mumps virus IgM, IgG titer, IgG avidity, neutralizing antibody titers of mumps patients Patient
Age
Gender
Vaccination status
IgM in acute sera
IgG (titer)a
P1/2001 P2/2001 P3/2002 P4/2002 P5/2002 P6/2002 P7/2002 P8/2002 P9/2002 P10/2002 P11/2002 P12/2003 P13/2003 P14/2003 P15/2003
21 16 22 20 18 20 19 18 22 18 19 20 26 18 18
M M F M M M M F M F M F M M M
Unknown Vaccinated 14 years ago Vaccinated 20 years ago Vaccinated 18 years ago Unknown Unknown Vaccinated 17 years ago Vaccinated 16 years ago Vaccinated 20 years ago Unknown Vaccinated 17 years ago Unknown Unvaccinated Vaccinated 14 years ago Vaccinated 16 years ago
+ + + + + + + ± ± ± + + + – +
1:3600/1:4800 1:1000/1:2600 1:1.400/1:3.600 neg/1:2400 neg/1:3200 1:2100/1:8200 1:1700/1:5900 1:3300/1:9600 1:1400/1:3000 1:2600/10700 neg/1:300 1:600/1:1300 1:200/1:1200 1:500/1:2000 1:1500/1:7500
AI in acute sera (%) 23 27 19 – – 26 45 24 19 22 – 18 31 44 17
PRN with strain L-3 (titer)a
PRN with strain H (titer)a
PRN with strain C (titer)a
1:4/1:4 1:4/1:4 n.d/1:4 n.d./1:4 n.d./1:4 n.d./1:4 1:8/1:8 n.d./1:8 n.d./1:8 n.d./1:4 n.d./1:4 1:4/1:8 1:4/1:4 1:8/1:8 n.d./1:8
n.d./1:4 n.d./1:8 n.d./1:4 n.d./1:8 n.d./1:8 n.d./1:4 n.d./1:8 n.d./1:16 n.d./1:16 n.d./1:4 n.d./1:4 n.d./1:8 n.d./1:4 n.d./1:4 n.d./1:16
n.d./n.d. n.d./1:4 n.d./n.d. n.d./1:4 n.d./1:4 n.d./1:4 n.d./n.d. n.d./1:4 n.d./1:4 n.d./1:4 n.d./n.d. n.d./1:8 n.d./1:4 n.d./n.d. n.d./1:4
All vaccinated here patients were immunized only once, as a mandatory second vaccination was introduced in Minsk in 1999. AI: avidity index; PRN: plaque reduction neutralization assay; n.d.: not detectable level. a Acute serum samples were taken at admission/second samples were taken at discharge. Paired samples were taken with the interval of 7–12 days.
H viruses were isolated in Novosibirsk, Russia [13,16]. A negative serum control was included in each plate of the tested samples. In all PRN-assays, no virus neutralizing activity was measured with the negative control sample. 2.2.3. Nucleotide sequence analysis RNA was extracted from saliva and sera and was purified using QIAGEN RNeasy mini kit (Qiagen, Germany). For identifying the specific virus strain responsible for infection, RT-PCR was performed on saliva samples using specific primers to amplify the viral SH gene as previously described [21]. The primers for RT-PCR were 5 -tcaacacaatatcaagta3 (pos. 2964–2981) and 5 -ttctgtgttgtattgtga-3 (pos. 3401–3418). The PCR products were excised from the gel and purified using QIAquick Gel Extraction Kit (Qiagen, Germany). The purified products were directly sequenced the primers 5 -atgatctcatcaggtac-3 (pos. 2988–3004) and 5 tcctaagtttgttctgg-3 (pos. 3384–3400). Nucleotide sequences were determined by the CEQ 2000 Dye Terminator Cycle Sequencing Kit (Beckman Coulter, USA) and Beckman CEQ 2000 XL DNA Analysis System (Beckman Coulter, USA) according to manufacturer’s instructions. Nucleotide alignments analyses were performed using Vector NTI v. 8.0 (InforMax, Bethesda, MD) software package. The MEGA package (version 3.1) was used to generate a phylogenetic tree using neighbor joining and Kimura two-parameter methods. The statistical significance of a particular tree topology was evaluated by bootstrap re-sampling of the sequences 1000 times. 2.2.4. Statistical analysis Mumps PRN titers were normalized by logarithmic transformation to calculate mean titers (MT). In all cases, sera that were below the level of detection (titer less than 1:4) were
arbitrarily assigned a titer of 1:2 (1 − log2 ) for the purpose of statistical analysis. Quantitative results were expressed as MT ± S.D. (standard deviation). The statistical significance between MT was tested. Statistical analysis was conducted using Student’s t- or Chi square-test. Statistical significance was defined at P ≤ 0.05. Pearson correlation coefficient (r) was also calculated. 2.3. Definitions Primary vaccine failure (PVF) was defined as a previously vaccinated subject with clinical mumps and evidence of a primary immune response (PIR): IgM positive/IgG negative or IgG positive with low avidity [22]. Secondary vaccine failure (SVF) was defined as a previously vaccinated subject with clinical mumps and evidence of a secondary immune response (SIR): high avidity IgG antibody in acute phase sera [18].
3. Results 3.1. Clinical manifestations and vaccination history Twenty-two adults were admitted to the Infectious Diseases Hospital in Minsk with a diagnosis of mumps, all presenting with parotitis. Paired serum samples were obtained from 15 of these patients. All subsequent studies described henceforth are limited to this subgroup of 15 patients. As summarized in Table 1, nine of these patients received one dose of mumps-containing vaccine 14–20 years earlier at 12–24 months of age. One patient had not been previously vaccinated and the vaccination status of the remaining five patients could not be confirmed. Ten patients had bilateral
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parotitis (67%) and five had unilateral parotitis (33%). The submaxillary salivary glands were involved in nine patients (60%). Pancreatitis was diagnosed in two patients (13.3%), P1 and P2. Orchitis/epidimoorchitis was found in three males (20%), P1, P2 and P12. Patients P7, P13 and P14 had subfebrile body temperature while others were febrile. Clinical symptoms of meningitis (headache, vomiting, and fever) were registered in five patients (33.3%), P4, P5, P6, P10, P11, however spinal taps were not performed, thus the diagnosis could not be confirmed. The duration of the clinical course of mumps infection was from 7 to 12 days. All patients recovered completely. 3.2. Serological results All patients were negative for acute EBV and parainfluenza viruses 1, 2, and 3 by ELISA. Serum levels of MuV-specific IgM and IgG titer, IgG avidity, neutralizing antibody titers are shown in Table 1. IgM was detected in 11 of the 15 patients upon hospital admission. One patient (P14) was IgM negative and the presence of IgM was equivocal in three patients (P8–P10). Three patients were IgG negative at admission (P4, P5 and P11) but seroconverted by hospital discharge. The increase in IgG titers of the remaining patients was diagnostically significant (>2.1-fold), except for patient P1, who possessed a very high initial IgG titer. There was no correlation between the appearance of IgM and IgG concentration, nor between IgM and IgG levels and vaccination status. According to the definitions of vaccine failure, of the nine patients for whom vaccination could be positively confirmed, seven were cases of PVF (P2–P4, P8, P9, P11 and P15) and 2 were cases of SVF (P7 and P14). Vaccine virus (L-3) neutralizing antibodies were only detected in three of the nine vaccinees (P2, P7 and P14) at admission. Interestingly, the L-3 neutralizing antibody titers did not rise by discharge for these three patients. Vaccine virus neutralizing titers were in general higher at discharge than at admission (1.5 ± 0.7 − log2 dilution versus 2.4 ± 0.5 − log2 dilution; P < 0.0009). There was a good correlation between the AIs and the neutralizing antibody titers against the L-3 vaccine strain in the acute sera, r = 0.71, P < 0.05. None of the patients, including those for whom vaccination status could not be confirmed, had detectable neutralizing antibody against a wild type mumps genotype H strain upon admission. By hospital discharge, all 15 patients had neutralizing antibody against this strain. Notably, a strain similar to the one used here was detected in saliva of all patients by RT-PCR. Mean neutralizing titers against the genotype H virus were greater than those against a genotype C virus strain also tested in this study (2.7 ± 0.8 − log2 dilution versus 1.7 ± 0.6 − log2 dilution, respectively; P < 0.0006). The correlation between the neutralizing titers against mumps virus genotypes H and C in patients at discharge was moderate (r = 0.53; P < 0.05).
Patients P7, P13 and P14 had the highest avidity indexes and neutralizing titers and had the mildest clinical course of mumps infection. Those with symptoms of meningitis (P4, P5, P6, P10 and P11), had no detectable neutralizing antibody titers at admission and tended to have the lowest IgG AI scores (14.8 ± 3.8 versus 26.7 ± 3.3, P < 0.05), and three of those five (P4, P5 and P11) were initially IgG negative. 3.3. Nucleotide sequence analysis The diagnosis of mumps in all 22 patients admitted to the hospital was confirmed by RT-PCR. However, only the acute patients’ saliva samples were RT-PCR positive, but not serum samples. In all cases, the amplified SH gene sequence was identical and belonged to genotype H, based on the established genotype classification system for mumps viruses [23]. RNA extraction and RT-PCR studies were done in different rooms using air barrier filter pipette tips, so contamination and false positive results are highly unlikely. The precise sequence of this wild type H virus was deposited in Genbank under accession numbers (one per year) DQ136174 (P1/2001), DQ136175 (P4/2002) and DQ250041 (P14/2003), respectively. A phylogenetic comparison of the SH gene sequence of the different genotype H MuV isolates around the world are shown in Fig. 1. Notably, the isolates obtained in Belarus differed from the H2 strain isolated from nearby Russia (PetroNov-Russia-2004). The genotype H clinical isolates obtained from patients in the present study were most similar to the H1 subgroup (Fig. 1) reported earlier [23]. However, all Belarusian sequences contained an L at position 30 instead of the S typical to the H1 group [23]. In addition, all isolates contained an S instead of P at position 6. The latter was not previously reported for genotype H viruses [24]. Thus, the Belarusian isolates formed a distinct cluster within the genotype H1 group.
4. Discussion Vaccine failures have been linked to mumps outbreaks in populations with high vaccine coverage [13–16,25]. The assessment of VF cases provides information useful for the improvement of mumps immunization strategy. The WHO strategies to achieve mumps elimination include not only very high coverage with the first dose of mumps vaccine, but also a second opportunity for vaccination and conducting catchup immunization of susceptible cohorts [17]. It is well known that VFs are less common in recipients of a two-dose regimen [14,26,27]. In all cases of mumps vaccine failure in our study, only one dose of vaccine was administered, and in most of the cases primary VF (7 from 9 vaccinated patients) was identified based on the AIs. It should be mentioned that different concentrations of urea used in mumps virus IgG
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Fig. 1. Genotype C sequences were included as an out-group. Phylogenetic analysis was performed using the Kimura two-parameter model as a model of nucleotide substitution and using the neighbor-joining method to reconstruct the phylogenetic tree (MEGA version 3.1 software package). The statistical significance of the phylogenies constructed was estimated by bootstrap analysis with 1000 pseudoreplicate data sets.
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avidity assay might affect assessment of secondary vaccine failure rate in outbreak settings [18,25]. Wherein, low avidity IgG signifies an immune response in immunologically naive patients, high avidity IgG signifies an immune response in patients with preexisting B-cell memory [28]. Notably, in contrast to conventional thinking, IgM can be detected in secondary immune responses (as we found in P7 here) and therefore does not serve as a marker of primary infection per se [25,29,30]. In many of our cases, patients possessed high mumps virus specific ELISA IgG titers in acute phase sera, yet avidity was low. The lack (or weak) association between IgG titers and their avidity have been reported earlier for a number of agents [31–34]. The PRN assay using the L-3 vaccine strain was invaluably helpful in the assessment of VF cases in our study. With one exception, all of the vaccinated patients assigned to the PVF-group lacked antibody neutralizing activity against the L-3 mumps vaccine strain despite being IgG-positive. At the same time, both patients assigned to the SVF-group demonstrated a pretty mature immune response based on the AIs as 44–45% which was also characterized by the PRN titer against L-3 vaccine strain as 1:8. Interestingly, at discharge the majority of patients demonstrated a rise in neutralizing titers against the L-3 strain independently of their vaccination history. The main finding here was that all patients independent of vaccination history, lacked neutralizing antibodies against the genotype H MuV, which is likely responsible for their susceptibility. In a recent publication, data was presented supporting the notion that sera possessing only low levels of mumps neutralizing antibody may not be broadly protective due to antigenic differences between MuV strains [35]. This is in line with reports by others proposing genotypespecific antibody responses [36,37]. In the present study, all acute phase sera possessing neutralizing antibody were of low titer and were capable of neutralizing the L-3 vaccine virus strain but not the wild type genotype H or C MuV strains tested. However, at discharge all sera demonstrated a MuV-specific neutralizing antibody response in that all convalescent sera could more effective neutralize a genotype MuV strain similar to the one detected by RT-PCR in patient saliva in comparison with a contemporary wild genotype MuV strain. This highlights the need for a more intensive examination of levels of neutralizing antibody required to confer broad cross-strain protection. Considering that mumps virus is serologically monotypic (despite antigenic difference between virus strains), such a “threshold” titer should exist. Our data indicate the importance of the assessment of avidity index and neutralizing antibody titers, against both the vaccine virus and the circulating viruses, for investigation of VF cases. The simplified dichotomic classification of vaccine failures (primary versus secondary) based only on the AIs may be problematic since a “grey zone” likely exists as was noted for measles by Paunio [38]. Earlier Narita et al. [39] also speculated that SVF comprises a wide variety of immune
responses to vaccine and that one extreme mimics PVF in terms of not only clinical presentation, but also serological response. The latter could take place in some of our patients. The PVF-group formed on the basis of AIs in our study consisted of seronegative subjects as well as seropositive subjects possessing IgG of low AIs, either lacking neutralizing antibody or possessing minimal titers of neutralizing antibody. Moreover, basing VF only on antibody data may be misleading and incomplete [40,41]. The reflexive focus on humoral immunity has overshadowed the role of the cellular response. Some of the mumps VF cases in our patients may be more clearly explained by the additional investigation of cellular mechanisms. For measles, individuals with undetectable or low levels of antibody may not necessarily be susceptible to infection, since cell-mediated immunity also affords protection [40,41]. Thus, it is of importance to develop a reliable laboratory protocol specifying the tests for investigation both cellular and humoral immune responses. The latter could shed much needed light on the issue of the VF and its classification. Genetic analysis is indispensable in understanding the epidemiology of mumps infection and in examining possible genotype-specific immunity in the population. Interestingly, the Belarus isolates belonged to a distinct cluster within the genotype H1 group of MuV. That this particular strain was isolated from all patients in our study over the 3-year period suggests that this strain is endemic to this geographic region. In conclusion, in all cases of vaccine failure identified in our study, only one dose of vaccine was administered and in most of these cases, primary VF was implicated based on the absence of markers suggestive of a mature and functional immune response. Whether or not the absence of sufficient titers of neutralizing antibody in response to vaccination or the absence of neutralizing antibodies specific for the endemic mumps virus strain were responsible for susceptibility of vaccinees to infection cannot be conclusively determined, but both are indicated. What is clear is that cause of vaccine failure is complex and multifactorial. It is therefore of high importance to develop a reliable laboratory protocol specifying all tests to be performed, analyzed and interpreted for VF case investigation. Our findings suggest that proper assessment of vaccine failure should include IgG avidity testing as well as a comprehensive assessment of the functional antibody response, i.e., the differential ability of serum antibody to neutralize both the vaccine virus as well as the endemic challenge virus strain.
Acknowledgements The work has been done in the framework of a grant BTEP ID#16, 2002. No official support or endorsement of this article by the Food and Drug Administration is intended or should be inferred. We thank our colleges Malisheva T. and D’achkova L. for the excellent technical assistance.
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